JP2019534036A - Adenovirus armed with a bispecific T cell engager (BiTE) - Google Patents

Abstract

A modified adenovirus armed with a bispecific T cell engager (BiTE), in particular enadenocileev (EnAd), comprising at least two binding domains, at least one of the domains Modified adenoviruses that are specific for surface antigens on T cells of interest. Also provided are viral formulations for treatment, such as in a pharmaceutical formulation comprising the virus, the use of the virus, and the treatment of cancer. The present disclosure also extends to methods for preparing viruses. [Selection] Figure 31

Description

  The present disclosure describes a modified adenovirus armed with a bispecific T cell engager (BiTE) comprising at least two binding domains, wherein at least one domain is specific for a surface antigen on a T cell of interest, in particular It relates to enadenotucirev (EnAd). The present disclosure further relates to compositions such as viruses, the use of viruses, and pharmaceutical formulations including viral formulations, particularly in the treatment of cancer, also in the treatment of cancer. The present disclosure also extends to methods for preparing viruses and DNA encoding them.

  Cancer is still a major social burden for society in terms of the difficulties and suffering of patients and their loved ones and also the high economic costs of treating, caring and supporting patients.

  A wide variety of therapies have been developed for the treatment of cancer, including chemotherapeutic agents, radiation therapy and most recently biologics such as antibodies. Antibody-based therapies for cancer have been established over the past 15 years and are now one of the most successful and important strategies for treating patients with hematological malignancies and solid tumors. Examples of monoclonal antibody-based anticancer therapies currently in clinical use include rituximab targeting CD20, bevacizumab targeting VEGF, cetuximab targeting EGFR, and lavetuzumab targeting CEA It is.

Among the various antibody formats developed, the bispecific T cell engager (BiTE) has shown many promises. They are specific to the CD3ε subunit of the T cell TCR complex and are also relatively simple bispecific molecules that target antigens of interest such as cancer antigens. Since BiTE is specific for TCR complexes, this indicates that BiTE activates resident T cells to kill cells that express specific target antigens on their cell surface, such as cancer cells. to enable. An important property of BiTE is the ability of CD4 ⁺ and non-activated CD8 ⁺ T cells to target cancer cells. In other words, BiTES activated T cells can cause cells to die regardless of MHC expression on the cell surface. This is important because some tumor cells down-regulate MHC, thereby making them resistant to drugs such as CAR-T cells and immTAC.

  Unfortunately, BiTE has poor circulation kinetics compared to full-length antibodies. This means that most BiTEs do not reach their target cells when administered to a patient. Furthermore, the use of high affinity anti-CD3 ScFv as part of BiTE can result in strong binding to T cells in the blood, which also interferes with delivery to the tumor. As a result, BiTE cannot reach their greatest potential as an anti-cancer therapy because they cannot be delivered effectively to tumor cells.

  The effects of therapeutic agents such as BiTE on tumor cells have become more apparent as solid tumors protect themselves in vivo in many ways, for example by developing a stroma around the tumor The need for effective delivery is becoming increasingly important. Progression to carcinoma is associated with the proliferation of epithelial cells (mitotic cells) along with the development of activated tumor stroma. In this case, since the turnover increases, extracellular matrix (ECM) components such as collagen bundles are decomposed. The number of inflammatory cells increases and fibroblasts differentiate into myofibroblasts, resulting in the expression of growth factors, matrix components and degrading proteases. Angiogenesis is maintained, resulting in numerous leaky tumor blood vessels. Following activation of the tumor stroma with persistent angiogenesis, invasion by tumor cells begins through the degraded basement membrane and blood vessels infiltrate the tumor tissue.

  This stroma is physical protection in that it can have the function of capturing immune cells that are sent to fight the tumor. In addition, the stroma shields the tumor's hypoxic microenvironment, which is tolerated and optimized for tumor growth. There are several theories that cells in the stroma are the energy source in the tumor.

  Most of the tumor stroma is fibroblasts, which are damaged to serve the purpose of cancer. Other cells that infiltrate the stroma are tumor-associated macrophages (TAM), a type 2 (M2) that can promote tumor growth by secreting cytokines and chemokines such as IL-10 that suppress immune responses. ) Macrophages.

  Targeting the tumor stroma is particularly difficult because the cells that form the environment are “natural” immune or connective tissue cells found throughout the body. Therefore, targeting these cells with therapeutic agents can have serious off-target effects.

  Therefore, there is a need for an improved method of delivering BiTE directly to tumor cells that can provide maximum therapeutic benefit, particularly delivery to tumor cells surrounded by stromal fibroblasts. ing.

  We have engineered one of the most effective methods for delivering therapeutic agents directly to tumors to activate T cells and express antigen targeting agents, such as in the stroma. Use oncolytic adenovirus.

Accordingly, the present disclosure provides formula (I):
5'ITR-B1-BA-B2-BX-BB-BY-B3-3'ITR (I)
Where
B1 is a bond or comprises E1A, E1B or E1A-E1B;
BA includes -E2B-L1-L2-L3-E2A-L4,
B2 is a bond or comprises E3;
BX is a bond or a DNA sequence containing a restriction site, one or more transgenes, or both;
BB includes L5,
BY is a DNA sequence comprising a binding or restriction site, one or more transgenes, or both;
B3 is a bond or comprises E4;
The adenovirus encodes a bispecific T cell engager (BiTE) comprising at least two binding domains, at least one of which is a surface antigen on a subject immune cell, such as a subject T cell. Specific to
Adenoviruses provide an adenovirus comprising a sequence of formula (I) which is EnAd or Ad11.

  One or more BiTEs according to the present disclosure do not contain a transmembrane domain and are therefore not expressed on the surface of cancer cells, but rather contain a signal sequence to facilitate the release of BiTE molecules from the cancer cells.

The following paragraphs are an overview of the present disclosure.
1. Formula (I):
5'ITR-B1-BA-B2-BX-BB-BY-B3-3'ITR (I)
Where
B1 is a bond or comprises E1A, E1B or E1A-E1B;
BA includes -E2B-L1-L2-L3-E2A-L4,
B2 is a bond or comprises E3;
BX is a bond or a DNA sequence containing a restriction site, one or more transgenes, or both;
BB includes L5,
BY is a DNA sequence comprising a binding or restriction site, one or more transgenes, or both;
B3 is a bond or comprises E4;
The adenovirus encodes a bispecific T cell engager (BiTE) comprising at least two binding domains, wherein at least one of the domains is specific for a surface antigen on a subject's immune cells, such as the subject's T cells And
Adenoviruses provide an adenovirus comprising a sequence of formula (I) which is EnAd or Ad11.
2. The adenovirus of paragraph 1, wherein the adenovirus is EnAd.
3. The adenovirus according to paragraph 1 or 2, wherein the surface antigen is a component of a T cell receptor complex (TCR) such as CD3, TCR-α and TCR-β.
4). Adenovirus according to paragraph 3, wherein the surface antigen is CD3, such as CD3-ε, CD3-γ, and CD3-δ, in particular CD3-ε.
5. One of the binding domains is CEA, MUC-1, EpCAM, HER receptor HER1, HER2, HER3, HER4, PEM, A33, G250, carbohydrate antigens Le ^y , Le ^X , Le ^b , PSMA, TAG-72, STEAP1, The adenovirus according to any one of paragraphs 1 to 4, which is specific for tumor antigens such as CD166, CD24, CD44, E-cadherin, SPARC, ErbB2 and ErbB3.
6). 6. The adenovirus of paragraph 5, wherein one of the binding domains is specific for EpCAM, eg, EpCAM comprising the amino acid sequence set forth in SEQ ID NO: 28.
7). One of the binding domains is a tumor stromal antigen such as fibroblast activation protein (FAP), TREM1, IGFBP7, FSP-1, platelet-derived growth factor-α receptor (PDGFR-α), platelet-derived growth factor— The adenovirus according to any one of paragraphs 1 to 4, which is specific for β receptor (PDGFR-β) and vimentin.
8). 8. The adenovirus of paragraph 7, wherein one of the binding domains is specific for FAP, eg, FAP comprising the amino acid sequence set forth in SEQ ID NO: 30.
9. The adenovirus according to paragraph 7 or 8, wherein the stromal antigen is an antigen and is selected from bone marrow-derived suppressor cell antigens, tumor-associated macrophages, and combinations thereof.
10. The adenovirus of paragraph 9, wherein the antigen is selected from CD163, CD206, CD68, CD11c, CD11b, CD14, CSF1 receptor, CD15, CD33, CD66b and combinations of two or more thereof.
11. The adenovirus according to any one of paragraphs 1-3 and 5-10, wherein one of the binding domains in BiTE is specific for a non-TCR activating protein such as CD31, CD2 and CD277.
12 The adenovirus according to any one of paragraphs 1 to 11, wherein at least one of BX and BY is not a bond.
13. The adenovirus according to any one of paragraphs 1 to 12, wherein the adenovirus is chimeric.
14 The adenovirus according to any one of paragraphs 1 to 13, wherein the adenovirus is oncolytic.
15. The adenovirus according to any one of paragraphs 1 to 14, which is capable of replicating adenovirus.
16. The adenovirus of paragraph 13, wherein the adenovirus is capable of replication.
17. The adenovirus according to any one of paragraphs 1 to 14, wherein the adenovirus is replication-defective.
18. The adenovirus according to any one of paragraphs 1 to 17, wherein BX comprises one or more transgenes or transgene cassettes.
19. The adenovirus according to any one of paragraphs 1-16, wherein BY comprises one or more transgenes or transgene cassettes.
20. The adenovirus according to any one of paragraphs 1-19, wherein the one or more transgenes or transgene cassettes are under the control of an endogenous or exogenous promoter such as an endogenous promoter.
21. 21. The adenovirus of paragraph 20, wherein the transgene or transgene cassette is under the control of an endogenous promoter selected from the group consisting of an E4 promoter and a major late promoter, particularly a major late promoter.
22. The adenovirus of paragraph 19, wherein the transgene or transgene cassette is under the control of an exogenous promoter such as CMV.
23. The transgene cassette is
a. Splice acceptor sequence,
b. An internal ribosome entry sequence or an A peptide with high self-cleavage efficiency,
c. Kozak sequence, and d. The adenovirus according to any one of paragraphs 1-22, further comprising a regulatory element selected independently from their combination.
24. 24. The adenovirus of paragraph 23, wherein the transgene cassette comprises a Kozak sequence at the start of the protein coding sequence.
25. 25. The adenovirus according to any one of claims 1 to 24, wherein the transgene cassette encodes a high self-cleaving efficiency A peptide.
26. The adenovirus according to any one of paragraphs 1-25, wherein the transgene cassette further comprises a polyadenylation sequence.
27. 27. The adenovirus according to any one of paragraphs 1 to 26, wherein the transgene cassette further comprises a restriction site at the “end” of the DNA sequence and / or at the “end” of the DNA sequence.
28. 28. Adenovirus according to any of paragraphs 1-27, wherein at least one transgene cassette encodes a monocistronic mRNA.
29. 29. Adenovirus according to any one of paragraphs 1 to 28, wherein BiTE has a short half-life, for example 48 hours or less.
30. The adenovirus according to any one of paragraphs 1 to 29, wherein BiTE is encoded in a region selected from E1, E3, BX, BY and combinations thereof.
31. The adenovirus of paragraph 30, wherein BiTE is encoded at least at position BX, for example under the control of a major late promoter.
32. 30. The adenovirus according to any one of paragraphs 1 to 29, wherein the adenovirus further encodes a second BiTE.
33. The first BiTE molecule is specific for a tumor antigen, eg, a tumor antigen (eg, as described herein), and the second BiTE molecule is a tumor stromal antigen, eg, a stromal antigen (eg, as described herein). The adenovirus of paragraph 32, which is specific for
34. 34. The adenovirus according to any one of paragraphs 1-33, wherein the adenovirus further comprises a cytokine or chemokine or an immunomodulator (such as a cytokine or chemokine).
35. Cytokines or chemokines are MIP1α, IL-1α, IL-1β, IL-6, IL-9, IL-12, IL-13, IL-17, IL-18, IL-22, IL-23, IL-24. IL-25, IL-26, IL-27, IL-33, IL-35, IL-2, IL-4, IL-5, IL-7, IL-10, IL-15, IL-21, IL -25, IL-1RA, IFNα, IFNβ, IFNγ, TNFα, lymphotoxin α (LTA), Flt3L, GM-CSF, IL-8, CCL2, CCL3, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13 , CXCL12, CCL2, CCL19, CCL21 (eg, IL-1α, IL-1β, IL-6, IL-9, IL-12, I -13, IL) -17, IL-18, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-33, IL-35, IL-2, IL- 4, IL-5, IL-7, IL-10, IL-15, IL-21, IL-25, IL-1RA, IFNα, IFNβ, IFNγ, TNFα, lymphotoxin α (LTA), GM-CSF, IL- 8, CCL2, CCL3, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19, CCL21), for example, IL-12, IL-18, IL-22, IL-7, IL- 15, etc., IL-21, IFNγ, TNFα, lymphotoxin α (LTA), CCL3, CCL5, CXCL9, CXCL10, C CL12, CCL2, CCL19 and is selected from CCL21, adenovirus according to paragraph 34.
36. Adenovirus is an immunomodulator such as an antibody or antibody fragment, or CTLA-4, PD-1, PD-L1, PD-L2, VISTA, B7-H3, B7-H4, HVEM, ILT-2, ILT-3 , ILT-4, TIM-3, LAG-3, BTLA, LIGHT or CD160, such as for CTLA-4, PD-1, PD-L1 and PD-L2, or CD28, CD80, CD86, CD83, ICOS, B7H2, 34. The adenovirus according to any one of paragraphs 1-35, further comprising a protein or peptide ligand specific for a checkpoint protein against costimulatory molecules such as TL1A and 4-1BB.
37. The adeno of any one of paragraphs 1-36, wherein BiTE comprises a VH domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 8, 13 or 18, or an amino acid sequence at least 95% identical thereto. Virus.
38. Paragraphs 1 to 37, wherein BiTE comprises a VL domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9, 12, or 17, or an amino acid sequence at least 95% identical thereto. Adenovirus.
39. Adenovirus according to any one of paragraphs 1 to 36, wherein BiTE comprises an scFv comprising the amino acid sequence set forth in any one of SEQ ID NOs: 7, 11 or 16, or an amino acid sequence at least 95% identical thereto. .
40. Paragraphs 1 to 39, wherein BiTE comprises the amino acid sequence set forth in SEQ ID NO: 2 or 4, or an amino acid sequence that is at least 95% identical thereto, eg, the amino acid sequence set forth in SEQ ID NO: 73 or 75 Adenovirus.
41. The adenovirus comprises a DNA sequence as set forth in any one of SEQ ID NOs: 34-37, or a DNA acid sequence that is at least 95% identical to this, eg, a DNA sequence as set forth in any one of SEQ ID NOs: 79-82 The adenovirus according to any one of paragraphs 1 to 40.
42. A composition comprising the adenovirus according to any one of paragraphs 1 to 41 and a diluent or carrier.
43. 43. A method of treating a patient, comprising administering a therapeutically effective amount of the adenovirus according to any one of paragraphs 1-41 or the composition of paragraph 42.
44. 44. The method of paragraph 43 for the treatment of cancer, particularly solid tumors.
In one embodiment, an adenovirus according to the present disclosure encodes at least one additional transgene, for example 1, 2, 3 or 4 additional transgenes.
In one embodiment, different cleavage peptides are encoded between each gene.
In one embodiment, all transgenes are in one place in the virus, eg, located in BY.

  Advantageously, we have armed the adenovirus with BiTE molecules, thereby allowing the bispecific antibody fragment molecules to “piggy” on the ability of the adenovirus to selectively infect cancer cells, thereby creating tumors. It has been discovered that it enables targeted delivery of BiTE to cells.

  Advantageously, BiTE is small and can be produced in mammalian cells. Thus, upon infection with an adenovirus of the present disclosure, BiTE molecules may be synthesized and secreted by tumor cells and act locally to spread beyond the virus's direct footprint. Thus, this allows BiTE to spread beyond the direct site of infection, but at the same time limits the spread of the virus too much beyond the nest of infected tumor cells. This minimizes the risk of undesirable off-target effects.

  In one embodiment, the adenovirus is EnAd. EnAd has been shown to have enhanced oncolytic activity compared to prior art adenoviruses. EnAd has also been shown to have high selectivity for human epithelial derived cancer cells such as colon cancer cells, lung cancer cells, bladder cancer cells and kidney cancer cells. T cells are activated by BiTE molecules to attack target cells, while EnAd can simultaneously infect and lyse cancer cells, making it an ideal delivery vehicle for BiTE molecules. This results in a two-sided attack on the tumor with a synergistic oncolytic effect.

  In one embodiment, the surface antigen is a component of a T cell receptor complex (TCR) such as CD3, TCR-α and TCR-β.

  In one embodiment, the surface antigen is a specific CD3ε, eg, CD3 such as CD3ε, CD3γ, and CD3δ.

In one embodiment, one of the binding domains is, for example, CEA, MUC-1, EpCAM, HER receptor (eg, HER1, HER2, HER3, HER4, etc.), PEM, A33, G250, carbohydrate antigens Le ^Y , Le ^x , Specific for tumor antigens such as Le ^b , PSMA, TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2 and ErbB3, especially EpCAM.

  In one embodiment, one of the binding domains is specific for an EpCAM such as an EpCAM comprising, for example, the amino acid sequence set forth in SEQ ID NO: 28 or a sequence that is at least 95% identical thereto.

  In one embodiment, one of the binding domains is, for example, fibroblast activation protein (FAP), TREM1, IGFBP7, FSP-1, platelet derived growth factor-α receptor (PDGFR-α), platelet derived growth factor Specific for tumor stromal antigens such as β receptor (PDGFR-β) and vimentin. Advantageously, stromal cells that express these antigens (non-transformed cells) do not undergo the same level of mutation resistance selection process as transformed cells. Therefore, these cells are not “moving targets” and are therefore easier to target for cancer therapy. Furthermore, the types of receptors found in stromal cells are often common across different types of cancer. Therefore, targeting one of the above antigens is likely to be effective for multiple cancer types.

  In one embodiment, one of the binding domains is specific for a FAP, such as, for example, an amino acid sequence set forth in SEQ ID NO: 30, or a FAP comprising a sequence that is at least 95% identical thereto. Advantageously, FAP is upregulated in tumor associated fibroblasts. Fibroblasts are an important component of solid cancer that supports growth, invasion, and recovery from intervention. They typically account for 40-60% of the cells in advanced cancer. Advantageously, fibroblasts are genetically stable cells that are less likely to escape treatment than cancer cells. Activated fibroblasts are also relatively similar across various types of tumors. Thus, by activating T cells and targeting and killing tumor-associated fibroblasts that express FAP, adenoviruses of the present disclosure are capable of immunosuppressive pathways such as those mediated by IL-10, TGFβ, and IDO. Can help reduce the spectrum of

  Other stromal targets include tumor associated macrophages and bone marrow derived suppressor cell antigens such as CD163, CD206, CD68, CD11c, CD11b, CD14, CSF1 receptor, CD15, CD33, CD66b and combinations of two or more thereof. It is done.

  In one embodiment, one of the binding domains within BiTE is specific for non-TCR activated proteins such as CD31, CD2, and CD277.

  In one embodiment, one of the binding domains is directed to a surface antigen on the T cell of the subject, such as selected from CD3 (such as CD3 delta, CD3 epsilon or CD3 gamma), TCR-α chain and TCR-β chain. Specific and one binding domain is specific for a tumor antigen.

In one embodiment, one of the binding domains is specific for CD3 and another binding domain is for example CEA, MUC-1, EpCAM, HER receptor HER1, HER2, HER3, HER4, PEM, A33, G250, Specific for a tumor antigen selected from the group consisting of carbohydrate antigens Le ^Y , Le ^X , Le ^b , PSMA, TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2 and ErbB3.

  In one embodiment, one of the binding domains is specific for CD3 (such as CD3 delta, CD3 epsilon or CD3 gamma) and another binding domain is specific for EpCAM.

  In one embodiment, one of the binding domains is specific for CD3ε and another binding domain is specific for EpCAM.

  In one embodiment, one of the binding domains is specific for a surface antigen on a T cell of interest, such as selected from CD3 (such as CD3 delta, CD3 epsilon or CD3 gamma), TCR-α and TCR-β. And another binding domain is specific for a tumor stromal antigen.

  In one embodiment, one of the binding domains is specific for CD3 (such as CD3 delta, CD3 epsilon or CD3 gamma) and another binding domain is, for example, fibroblast activation protein (FAP), TREM1, Specific to tumor stromal antigen selected from the group consisting of IGFBP7, FSP-1, platelet derived growth factor-α receptor (PDGFR-α), platelet derived growth factor-β receptor (PDGFR-β) and vimentin It is.

  In one embodiment, one of the binding domains is specific for CD3 (such as CD3 delta, CD3 epsilon or CD3 gamma) and another binding domain is specific for FAP.

  In one embodiment, one of the binding domains is specific for CD3ε and another binding domain is specific for FAP.

  In one embodiment, one of the binding domains is specific for a surface antigen on the T cell of interest, such as CD3, TCR-α and TCR-β, and another binding domain is specific for a non-TCR activated protein. It is.

  In one embodiment, one of the binding domains is specific for CD3 (such as CD3 delta, CD3 epsilon or CD3 gamma) and the other binding domain is a non-TCR activation selected from the group consisting of CD31, CD2 and CD277. Specific for proteins.

  In one embodiment, one of the binding domains is specific for CD3ε and another binding domain is specific for a non-TCR activating protein selected from the group consisting of CD31, CD2 and CD277.

  In one embodiment, at least one of BX or BY is not a bond.

  In one embodiment, BX is not a bond.

  In one embodiment, BY is not a bond.

  In one embodiment, both BX and BY are not bonds.

  In one embodiment, the adenovirus is chimeric.

  In one embodiment, the adenovirus is oncolytic.

  In one embodiment, the adenovirus is chimeric and oncolytic.

  In one embodiment, the adenovirus is replicable.

  In one embodiment, the adenovirus is chimeric, oncolytic and replicable.

  In one embodiment, the adenovirus is capable of replication.

  In another embodiment, the adenovirus is chimeric, oncolytic, and capable of replication.

  In one embodiment, the adenovirus is replication-defective, ie a vector.

  In one embodiment, BX comprises a transgene or transgene cassette, in particular a transgene cassette encoding BiTE according to the present disclosure.

  In one embodiment, BY comprises a transgene or transgene cassette, in particular a transgene cassette encoding BiTE according to the present disclosure.

  In one embodiment, BY comprises a transgene or transgene cassette, in particular a transgene cassette encoding BiTE according to the present disclosure, and BX represents binding.

  In one embodiment, both BX and BY contain a transgene or transgene cassette.

  In one embodiment, the one or more transgenes or transgene cassettes are under the control of an endogenous or exogenous promoter, such as an endogenous promoter. Advantageously, when under the control of these promoters, the virus remains capable of replicating and can express BiTE and / or other proteins. Thus, the selected BiTE is expressed by cancer cells. The use of an exogenous promoter can be advantageous in some embodiments because it can strongly and constitutively express an antibody or fragment, which in some situations can be very It can be particularly useful when having a wide range of cancers. Using an endogenous promoter can be advantageous because it reduces the size of the transgene cassette that needs to be incorporated to express BiTE, ie, the cassette does not need to contain an exogenous promoter. Can be smaller.

  Thus, in one embodiment, the transgene or transgene cassette is under the control of an endogenous promoter selected from the group consisting of E4 and the major late promoter, particularly the major late promoter. In contrast to constitutive exogenous promoters that continuously transcribe the transgene and may lead to inappropriate concentrations of antibodies or fragments, the transgene is only when the virus is replicating in cancer cells It can also be advantageous in the therapeutic context to use an endogenous promoter in the virus since it is expressed.

  In one embodiment, the transgene or transgene cassette (eg, encoding BiTE) is under the control of an exogenous promoter such as CMV. Advantageously, the use of a constitutive exogenous promoter results in continuous transcription of the transgene, which may be desirable in some cases.

  In one embodiment, one transgene or transgene cassette (eg, encoding BiTE) is under the control of an endogenous promoter and another transgene or transgene cassette (eg, encoding BiTE) is exogenous. It is under the control of a promoter.

  In one embodiment, all of the transgene or transgene cassette (eg, encoding BiTE) in the virus is under the control of an endogenous promoter.

  In another embodiment, all of the transgene or transgene cassette (eg, encoding BiTE) in the virus is under the control of an exogenous promoter.

In one embodiment, the transgene or transgene cassette is
i) splice acceptor sequence,
ii) an internal ribosome entry sequence or a high self-cleavage efficiency 2A peptide;
iii) further comprising regulatory elements selected independently from Kozak sequences, and iv) combinations thereof.
Thus, in one embodiment, the transgene cassette comprises i) or ii) or iii) or iv).

  In one embodiment, the transgene cassette is i) and ii), or i) and iii), or i) and iv), or ii) and iii), or ii) and iv), or iii) and iv). including.

  In one embodiment, the transgene cassette comprises i) and ii) and iii), or i) and ii) and iv), or i) and iii) and iv), or ii) and iii) and iv). .

  In one embodiment, the transgene cassette comprises i) and ii) and iii) and iv).

  In one embodiment, the transgene cassette includes a Kozak sequence at the start of a protein (eg, BiTE) coding sequence that assists in translation of the mRNA.

  In one embodiment, the transgene cassette encodes a high self-cleaving efficiency 2A peptide.

  In one embodiment, the transgene cassette further comprises a polyadenylation sequence.

  In one embodiment, the transgene cassette further comprises restriction sites at the 3 'end of the DNA sequence and / or at the 5' end of the DNA sequence.

  In one embodiment, at least one transgene cassette encodes a monocistronic mRNA.

  In one embodiment, the BiTE molecule has a short half-life, eg, 48 hours or less.

  In one embodiment, the BiTE molecule is encoded in a region selected from E1, E3, BX, BY and combinations thereof. Advantageously, we can insert various transgenes into BX and / or BY under the control of exogenous or endogenous promoters without adversely affecting the viral life cycle or vector stability. Established.

  In one embodiment, the BiTE molecule is encoded at least at position BX, for example under the control of the major late promoter. Advantageously, the transgene or transgene cassette allows BiTE or any additional molecule to be expressed together with the adenovirus itself. Importantly, the inventors have successfully demonstrated that BiTE expression does not significantly affect the replication ability of EnAd and does not adversely affect its oncolytic activity.

  In one embodiment, the BiTE molecule is encoded at least in the BY position, for example under the control of the major late promoter. Advantageously, the transgene or transgene cassette allows BiTE or any additional molecule to be expressed together with the adenovirus itself. Importantly, the inventors have successfully demonstrated that BiTE expression does not significantly affect the replication ability of EnAd and does not adversely affect its oncolytic activity.

  In one embodiment, the adenovirus further encodes a second BiTE.

  In one embodiment, the first BiTE molecule is specific for a tumor antigen, eg, a tumor antigen as described above, and the second BiTE molecule is specific for a tumor stromal antigen, eg, a stromal antigen as described above. is there.

In one embodiment, the first BiTE molecule is CEA, MUC-1, EpCAM, HER receptors HER1, HER2, HER3, HER4, PEM, A33, G250, carbohydrate antigen Le ^y , Le ^x , Le ^b , PSMA, Specific for a tumor antigen selected from the group consisting of TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2 and ErbB3, wherein the second BiTE molecule is a fibroblast activation protein ( A tumor stroma selected from the group consisting of FAP), TREM1, IGFBP7, FSP-1, platelet-derived growth factor-α receptor (PDGFR-α), platelet-derived growth factor-β receptor (PDGFR-β) and vimentin Specific for antigen.

  In one embodiment, the first BiTE molecule is specific for EpCAM and the second BiTE molecule is fibroblast activation protein (FAP), TREM1, IGFBP7, FSP-1, platelet derived growth factor-α receptor Specific to a tumor stromal antigen selected from the group consisting of a body (PDGFR-α), a platelet derived growth factor-β receptor (PDGFR-β) and vimentin.

In one embodiment, the first BiTE molecule is CEA, MUC-1, EpCAM, HER receptors HER1, HER2, HER3, HER4, PEM, A33, G250, carbohydrate antigen Le ^y , Le ^x , Le ^b , PSMA, Specific for a tumor antigen selected from the group consisting of TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2 and ErbB3, the second BiTE is specific for FAP.

  In one embodiment, the first BiTE molecule is specific for EpCAM and the second BiTE molecule is specific for FAP.

  In another embodiment, the first BiTE molecule is specific for a tumor antigen and the second BiTE molecule is specific for a non-TCR activated protein.

In one embodiment, the first BiTE molecule is CEA, MUC-1, EpCAM, HER receptors HER1, HER2, HER3, HER4, PEM, A33, G250, carbohydrate antigen Le ^y , Le ^x , Le ^b , PSMA, Specific for a tumor antigen selected from the group consisting of TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2 and ErbB3.

  In one embodiment, the first BiTE molecule is specific for EpCAM and the second BiTE molecule is specific for a non-TCR activated protein selected from the group consisting of CD31, CD2, and CD277.

  In one embodiment, the first BiTE molecule is specific for a tumor stromal antigen and the second BiTE molecule is specific for a non-TCR activated protein.

  In one embodiment, the first BiTE molecule is fibroblast activation protein (FAP), TREM1, IGFBP7, FSP-1, platelet derived growth factor-α receptor (PDGFR-α), platelet derived growth factor-β. A non-TCR activated protein that is specific for a tumor stroma antigen selected from the group consisting of a receptor (PDGFR-β) and vimentin, and wherein the second BiTe molecule is selected from the group consisting of CD31, CD2 and CD277 Specific.

  In one embodiment, the first BiTE molecule is specific for FAP and the second BiTE molecule is specific for a non-TCR activated protein selected from the group consisting of CD31, CD2, and CD277.

  In one embodiment, the adenovirus contains only one BiTE.

  In another embodiment, the adenovirus comprises two BiTEs.

  In another embodiment, the adenovirus comprises 3 BiTEs.

  In addition to encoding one, two or three BiTEs, the virus can also encode one, two, three or four additional transgenes.

  In one embodiment, the adenovirus further encodes a cytokine or chemokine.

  In one embodiment, the adenovirus further encodes a cytokine.

  In one embodiment, the adenovirus further encodes a chemokine.

  In another embodiment, the adenovirus further encodes cytokines and chemokines.

  In one embodiment, the adenovirus is independent of one BiTE and at least one cytokine or chemokine, eg 1, 2 or 3 cytokines, 1, 2 or 3 chemokines, or each gene independently of a chemokine cytokine. A combination of 2 or 3 genes encoding

  In another embodiment, the adenovirus comprises two BITEs and at least one cytokine or chemokine, such as 1 or 2 cytokines, 1 or 2 chemokines, or a combination of cytokines and chemokines.

  In another embodiment, the adenovirus comprises 3 BiTEs and at least one cytokine or chemokine.

  In one embodiment, the cytokine or chemokine is IL-1α, IL-1β, IL-6, IL-9, IL-12, IL-13, IL-17, IL-18, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-33, IL-35, IL-2, IL-4, IL-5, IL-7, IL-10, IL-15, IL- 21, IL-25, IL-1RA, IFNα, IFNβ, IFNγ, TNFα, lymphotoxin α (LTA) and GM-CSF, IL-8, CCL2, CCL3, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19, CCL21, such as IL-12, IL-18, IL-22, IL-7, IL-15, IL-2 , IFNγ, TNFα, lymphotoxin alpha (LTA), is selected from CCL3, CCL5, CXCL9, CXCL12, CCL2, CCL19, and CCL21.

  In one embodiment, the encoded cytokine is a TNF alpha superfamily (TNFRSF is TNF-alpha, TNF-C, OX40L, CD154, FasL, LIGHT, TL1A, CD70, Siva, CD153, 4-1BB ligand, TRAIL, RANKL, TWEAK, APRIL, BAFF, CAMLG, NGF, BDNF, NT-3, NT-4, GITR ligand, EDA-A, EDA-A2), TGF-beta superfamily, IL-1 family (ie IL- 1 and IL-8), IL-2 family, IL-10 family, IL-17 family, interferon family.

  In one embodiment, the chemokine is selected from the group comprising MIP-1 alpha, RANTES, IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19 and CCL21.

  In one embodiment, the adenovirus further comprises an antibody or antibody fragment specific for a checkpoint protein or costimulatory molecule, or an immunomodulator such as a protein or peptide ligand, or a specific binding ligand for such molecule.

  In one embodiment, the immunomodulator is CTLA-4, PD-1, PD-L1, PD-L2, VISTA, B7-H3, B7-H4, HVEM, ILT-2, ILT-3, ILT-4, An antibody or antibody fragment specific for a checkpoint protein such as TIM-3, LAG-3, BTLA, LIGHT or CD160, eg CTLA-4, PD-1, PD-L1 and PD-L2, or a protein or peptide ligand is there.

  In one embodiment, the immunomodulator is an inhibitor, such as a checkpoint inhibitor.

  In one embodiment, the immunomodulator is an agonist.

  In another embodiment, the immunomodulating agent is an antibody or antibody fragment specific for a costimulatory molecule such as CD28, CD80, CD86, CD83, ICOS, B7H2, TL1A and 4-1BB, or a protein or peptide ligand.

  In one embodiment, the adenovirus comprises a first antibody, antibody fragment, protein or peptide ligand specific for a checkpoint protein, and a second antibody, antibody fragment, protein or peptide ligand specific for a costimulatory molecule. Including.

  In one embodiment, BiTE comprises a VH domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 8, 13 or 18, or an amino acid sequence at least 95% identical thereto.

  In one embodiment, BiTE comprises a VL domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9, 12, or 17, or an amino acid sequence at least 95% identical thereto.

  In one embodiment, the BiTE comprises an scFv comprising the amino acid sequence set forth in any one of SEQ ID NOs: 7, 11, or 16, or an amino acid sequence that is at least 95% identical thereto.

  In one embodiment, BiTE (ie, encoded by adenovirus) for use in the present disclosure is a VH domain having the sequence set forth in SEQ ID NO: 8 or a sequence at least 95% identical thereto, and the sequence set forth in SEQ ID NO: 9 or A binding domain having a VL domain having a sequence at least 95% identical thereto.

  In one embodiment, BiTE (ie, encoded by adenovirus) for use in the present disclosure is a VH domain having a sequence set forth in SEQ ID NO: 13 or a sequence at least 95% identical thereto, and a sequence set forth in SEQ ID NO: 12 or A binding domain having a VL domain having a sequence at least 95% identical thereto.

  In one embodiment, BiTE (ie, encoded by adenovirus) for use in the present disclosure is a VH domain having the sequence set forth in SEQ ID NO: 18 or a sequence at least 95% identical thereto, and the sequence set forth in SEQ ID NO: 17, or A binding domain having a VL domain having a sequence at least 95% identical thereto.

  In one embodiment, BiTE (ie, encoded by adenovirus) for use in the present disclosure is a VH domain having the sequence set forth in SEQ ID NO: 8 or a sequence at least 95% identical thereto, and the sequence set forth in SEQ ID NO: 9 or A binding domain having a VL domain having a sequence at least 95% identical thereto, and a VH domain having the sequence shown in SEQ ID NO: 13 or at least 95% identical thereto, and the sequence shown in SEQ ID NO: 12 or at least 95% identical thereto A binding domain having a VL domain having a sequence.

  In one embodiment, BiTE (ie, encoded by adenovirus) for use in the present disclosure is a VH domain having the sequence set forth in SEQ ID NO: 8 or a sequence at least 95% identical thereto, and the sequence set forth in SEQ ID NO: 9 or A binding domain having a VL domain having a sequence at least 95% identical thereto, and a VH domain having a sequence shown in SEQ ID NO: 18 or at least 95% identical thereto, and a sequence shown in SEQ ID NO: 17 or at least 95% identical thereto A binding domain having a VL domain having a sequence.

  In one embodiment, BiTE (ie, encoded by adenovirus) for use in the present disclosure comprises a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 7, 11, 16, or any one thereof.

  In one embodiment, BiTE (ie, encoded by adenovirus) for use in the present disclosure comprises the sequence set forth in SEQ ID NO: 7 or a sequence that is at least 95% identical thereto.

  In one embodiment, BiTE (ie, encoded by adenovirus) for use in the present disclosure comprises the sequence set forth in SEQ ID NO: 11 or a sequence that is at least 95% identical thereto.

  In one embodiment, BiTE (ie, encoded by adenovirus) for use in the present disclosure comprises the sequence set forth in SEQ ID NO: 16 or a sequence that is at least 95% identical thereto.

  In one embodiment, BiTE comprises the amino acid sequence set forth in SEQ ID NO: 2 or 4, or an amino acid sequence that is at least 95% identical thereto, eg, the amino acid set forth in SEQ ID NO: 73 or 75.

  In one embodiment, an adenovirus according to the present disclosure comprises the DNA sequence set forth in any one of SEQ ID NOs: 34-37, or a DNA acid sequence that hybridizes under stringent conditions thereto.

  In one embodiment, an adenovirus according to the present disclosure comprises the DNA sequence set forth in any one of SEQ ID NOs: 79-82.

  In one embodiment, the adenovirus according to the present disclosure is any of SEQ ID NOs: 34, 35, 36, 37, 79, 80, 82, 96, 97, 98, 99, 100, 101, 102, 103, 120, 298. A DNA sequence shown in one, or a sequence encoding the same virus, or a sequence that hybridizes to any one of them under stringent conditions.

  In one embodiment, the adenovirus according to the present disclosure is any of SEQ ID NOs: 34, 35, 36, 37, 79, 80, 82, 96, 97, 98, 99, 100, 101, 102, 103, 120 or 298. Contains the DNA sequence shown in one.

  One skilled in the art recognizes that the DNA code is dysfunctional, and thus the present disclosure extends to EnAd or Ad11 encoding BiTE having the amino acids disclosed herein.

  The C-terminal Deca-His (HHHHHHHHHH, SEQ ID NO: 24) affinity tag is useful for the purification of BiTE or adenovirus. However, this is optional and may be excluded, for example, in the final product. Those skilled in the art will also recognize that other affinity tags other than Deca-His can be used and can be excluded without affecting the biological function of BiTE or adenovirus as well. Let's go. Thus, in one embodiment, BiTE comprises the amino acid sequence set forth in any one of SEQ ID NO: 2 or 4, but excludes a deca-His affinity tag at the C-terminus of the sequence set forth in SEQ ID NO: 73 or 75, for example. To do. In another embodiment, the adenovirus comprises a DNA sequence as set forth in any one of SEQ ID NOs: 34-37, eg, a deca-type such as the DNA sequence set forth in any one of SEQ ID NOs: 79-82. The His affinity tag is excluded.

  The exclusion of the deca-His affinity tag extends further to all other sequences disclosed herein that include a deca-His affinity tag, i.e., the present disclosure includes the same amino acid or The DNA sequence (HHHHHHHHHH or CATCACCCATCACCCATCACCACCCATCCATCAT) is included, for example as described in any one of SEQ ID NOs: 72-82.

  In one embodiment, the BiTE encoded by the virus of the present disclosure is under the control of an exogenous promoter, such as the CMV promoter. Exogenous can be placed between the MPL and the encoded transgene when the transgene is between the L5 region and the E4 region.

  Exogenous can be placed between the encoded transgene and L5 when the transgene is between the L5 and E3 regions.

  In one aspect, a composition comprising an adenovirus described herein and a diluent or carrier is provided.

  In one aspect, a method of treating a patient is provided comprising administering a therapeutically effective amount of an adenovirus or composition described herein.

  In one embodiment, the method is for the treatment of cancer, eg, epithelial cancer, particularly solid tumors.

  In one embodiment, there is provided a therapeutic method, particularly wherein the checkpoint inhibitor encodes a virus, comprising administering a virus according to the present disclosure in combination with a checkpoint inhibitor (eg, PD-1 or PDL1 inhibitor). The

  In one embodiment, a method of treatment comprising administering a virus according to the present disclosure not in combination with a checkpoint inhibitor (eg, those described elsewhere herein, such as PD-1 or PDL1 inhibitors). Provided, particularly where the checkpoint inhibitor does not encode a virus.

  BiTE encoded by the virus according to the present disclosure has the ability to enhance the cytotoxicity of the virus.

  Surprisingly, BiTes encoded by the virus according to the present disclosure activates even cells in the tumor suppressor environment, including CD4 + cells and / or CD8 + cells, eg, T cells in a fluid environment such as tumor ascites can do.

  Advantageously, BiTes encoded by the virus according to the present disclosure activates even T cells in a tumor suppressive environment, including cytotoxic T cells, eg, T cells in a fluid environment such as tumor ascites Can do.

  Even more surprising, BiTE encoded by the virus according to the present disclosure can stimulate (activate) T cell proliferation.

  Viruses encoding BiTE according to the present disclosure appear to be able to avoid, overcome or reverse the tumor immunosuppressive microenvironment.

  In one embodiment, activation of T cells results in upregulation of T cell markers, such as CD25.

  In one embodiment, the binding of BiTE in a virus according to the present disclosure is specific for a nascent antigen.

  The present disclosure also extends to the novel sequences disclosed herein.

  Immune cells as used herein include (but are not limited to) lymphocytes such as macrophages, neutrophils, dendritic cells, NK cells, T lymphocytes (particularly T cells and NKT cells), A cell that has a functional role in the immune system.

  The term antibody as used herein refers to a carbohydrate, polynucleotide, lipid, polypeptide via at least one antigen recognition site (also referred to herein as a binding site) located in the variable region of an immunoglobulin molecule. , Refers to an immunoglobulin molecule capable of specifically binding to a target antigen such as a peptide. Unless stated otherwise, the term extends to full-length antibodies and multispecific antibody molecules including full-length antibodies.

  As used herein, “antibody molecule” includes antibodies and binding fragments thereof, as well as any one multispecific format thereof.

  As used herein, an antigen binding site refers to a portion of a molecule comprising a pair of variable regions, particularly cognate pairs that specifically interact with a target antigen.

  Specifically, as used herein, a binding site that recognizes only the antigen for which it is specific, or an antigen that is specific compared to its affinity for an antigen that is non-specific Is intended to refer to a binding site having a significantly higher binding affinity, for example 5, 6, 7, 8, 9, 10 times higher binding affinity.

As used herein, binding fragments or antibody binding fragments include, but are not limited to, antibody binding fragments, Fab, modified Fab, Fab ′, modified Fab ′, F (ab ′) ₂ , Fv, single domain Multispecific antibody molecules comprising antibody binding fragments, including antibodies, scFv, bivalent, trivalent or tetravalent antibodies, Bis-scFv, diabody, triabodies, tetrabodies and any epitope binding fragment of any of the above (See, for example, Holliger and Hudson, 2005, Nature Biotech. 23 (9): 1126 to 1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2 (3), 209-217). Methods for making and producing these antibody fragments are well known in the art (see, eg, Verma et al., 1998, Journal of Immunological Methods, 216: 165-181). Other antibody fragments for use in the present disclosure include the Fab and Fab ′ fragments described in International Patent Applications WO05 / 003169, WO05 / 003170 and WO05 / 003171. Multivalent antibodies can include multispecificity, such as bispecificity, or can be monospecific (see, eg, WO92 / 22853 and WO05 / 113605).

  In one embodiment, the adenovirus comprises a multispecific antibody molecule.

  As used herein, a multispecific antibody molecule comprises two or more antigen binding domains, such as two (bispecific) or three (trispecific) or four (tetraspecific) binding domains. An antibody molecule having

The multispecific antibody molecules of the present disclosure can be constructed from various antibody fragments such as those described above. For example, a diabody is a bispecific antibody molecule consisting of a noncovalent dimer of ScFv fragments, while F (ab ′) ₂ is a bispecific consisting of two Fab fragments joined by a hinge region. It is an antibody molecule. Accordingly, those skilled in the art will recognize that different antibody fragments can be arranged in various combinations to produce bi- or multispecific antibody molecules.

Examples of trispecific or tetraspecific antibody formats include Fab ₃ , triabodies, tetrabodies, tribodies, DVD-Ig, IgG-scFv, ScFv ₂ -Fc, tandAb and DNL-Fab ₃ It is not limited to these.

  As used herein, a bispecific antibody molecule refers to a molecule having two antigen-binding domains that can bind to the same or different antigens. BiTE is a subclass of bispecific antibody molecules.

  Domains may bind to different antigens.

  Alternatively, the domains can all bind to the same antigen, including binding to the same epitope on the antigen, or binding to different epitopes on the same antigen.

Examples of bispecific antibody formats include bispecific T cell engager (BiTE), F (ab ') ₂ , F (ab')-ScFv2, di-scFv, diabody, minibody, scFv- Fc, DART, TandAb, ScDiabody, ScDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, ScFv-CH3 KIH (knobs in holes), Fab-ScFv-SCFv-CH , ScFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, intrabody, dock-and-lock antibody, ImmTAC, HSAbody, ScDiabody-HAS, human body and Tandem ScFv toxicity includes, but is not limited to, see, eg, Christoph Spiss et al., Molecular Immunology 67 (2015) pages 95-106.

  The adenovirus of the present disclosure comprises BiTE specific for at least a surface antigen on a T cell of interest. Examples of T cell surface antigens include CD3, CD2, VLA-1, CD8, CD4, CCR6, CXCR5, CD25, CD31, CD45RO, CD197, CD127, CD38, CD27, CD196, CD277 and CXCR3, particularly CD2, CD3, CD31 and CD277 are included, but not limited to these.

  As used herein, a bispecific T cell engager (BiTE) is an artificial bispecific monoclonal comprising amino acid sequences from 2 scFv of different antibodies or four different genes on a single peptide chain of about 55 KDa. Refers to the antibody class. One of the scFvs is specific for immune cells such as T cell antigens such as the CD3 receptor expressed on the surface of T cells. Other scFvs typically bind to tumor cells via tumor specific molecules. Thus, BiTE can form a link between T cells and tumor cells, thanks to their specificity for antigens on T cells and antigens on tumor cells. This leads to T cell activation and induces T cells to exert their cytotoxic effects on tumor cells, independent of MHC I or costimulatory molecules. Examples of BITE-based therapies currently approved or undergoing clinical trials include, for example, Blinatumomab (Blyncyto ™) and EpCAM that target CD19 and treat non-Hodgkin lymphoma and acute lymphoblastic leukemia And solitomab to treat gastrointestinal and lung cancer.

  In one embodiment, the immune cell engager (such as a T cell engager) is arranged, for example, in the format of VL1-linker 1-VH1-linker 2-VH2-linker 3-VL2, eg, a linker disclosed herein. A linker is used that is selected independently of the sequence.

  In one embodiment, the linker in BiTE according to the present disclosure is independently selected from SEQ ID NOs: 10, 14, 23, 124-162, and 166-297.

  In one embodiment, linker 1 and linker 3 have the same sequence as shown for example in any one of SEQ ID NOs: 10, 14, 23, 124-162 and 166-296, in particular 10, 14, and 23. .

  In one embodiment, linker 1 and linker 3 have different amino acid sequences independently selected from, for example, SEQ ID NOs: 10, 14, 23, 124-162 and 166-296, in particular 10, 14, and 23.

  In one embodiment, linker 1 is SEQ ID NO: 10.

  In one embodiment, linker 1 is SEQ ID NO: 14.

  In one embodiment, linker 3 is SEQ ID NO: 10.

  In one embodiment, linker 3 is SEQ ID NO: 14.

  In one embodiment, linker 1 and linker 3 are SEQ ID NO: 10.

  In one embodiment, linker 1 and linker 3 are SEQ ID NO: 14.

  In one embodiment, linker 1 is SEQ ID NO: 10 and linker 3 is SEQ ID NO: 14.

  In one embodiment, linker 1 is SEQ ID NO: 14 and linker 3 is SEQ ID NO: 10.

  In one embodiment, linker 2 is different from both linker 1 and linker 3.

  In one embodiment, linker 2 is selected from any one of SEQ ID NOs: 10, 14, 23, 124-162, and 166-297, such as SEQ ID NO: 297.

  In one embodiment, the linker 1 is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. Etc., within the range of 10-30 amino acids in length.

  In one embodiment, the linker 3 is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. Etc., within the range of 10-30 amino acids in length.

  In one embodiment, linker 2 is in the range of 2-10 amino acids long, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10.

  In one embodiment, VH1 and VL1 are specific for a T cell antigen according to the present disclosure, eg, CD3.

  In one embodiment, VH2 and VL2 are specific for immune cell antigens, such as T cell antigens such as CD3, according to the present disclosure.

  In one embodiment, VH1 and VL1 are specific for an antigen or subject, such as a cancer antigen or stromal antigen.

  In one embodiment, VH2 and VL2 are specific for an antigen or subject, such as a cancer antigen or stromal antigen.

  As used herein, the stroma or stromal antigen is on the molecular structure of the stromal matrix, eg, fibroblasts, tumor associated macrophages, dendritic cells, NK cells and / or T cells infiltrating the stroma. It refers to an antigen therapeutic target within the stroma, including those expressed in the molecular structure of the stromal matrix, such as connective tissue molecules or molecules associated with this matrix or antigens associated with cellular components of the stroma. Examples of stromal antigens include FAP, TGFβ, TREM1, IGFBP7, FSP-1, fibroblast-related antigen, NG2, endosialin (CD248), platelet-derived growth factor-α receptor (PDGFR-α), platelet-derived growth Factor-β receptor (PDGFR-β) and vimentin include but are not limited to.

  Fibroblasts can be targeted by using antibodies specific for antigen fibroblast activation protein (FAP), in particular FAP that does not bind to CD26 (see US2012 / 0258119, incorporated herein by reference). )

  FAP was initially identified as a serine protease on reactive stromal fibroblasts. Subsequent molecular cloning revealed that FAP is identical to seprase, a 170 kDa membrane-bound gelatinase expressed by the melanoma cell line. The full-length cDNA contains a 760 amino acid (aa) H-type transmembrane protease highly homologous to dipeptidyl peptidase IV (DPPIV) with 52% amino acid identity and almost 70% identity in the catalytic domain over the entire sequence. I was coding. US Pat. No. 5,587,299, incorporated herein by reference, describes nucleic acid molecules encoding FAP and uses thereof.

  FAP and DPPIV have similar gene sizes and are chromosomally adjacent to each other at 2q24, suggesting a gene duplication event (Genebank accession number U09278). Both proteins are members of the prolyl peptidase family. This class of enzymes is inducible, is active on the cell surface or in extracellular fluid, and can independently cleave N-terminal dipeptides from polypeptides having proline or alanine in the penultimate position. . DPPIV, also called CD26, is constitutively expressed by several cell types including fibroblasts, endothelial and epithelial cells, leukocyte subsets such as NK cells, T lymphocytes and macrophages. A small amount of DPPIV circulates as soluble protein in the blood. In contrast to DPPIV, FAP is usually not expressed in normal adult tissues, and its proteolytically active soluble form is called a2-antiplasmin cleaving enzyme (APCE). Significant FAP expression occurs in conditions associated with activated stroma, including wound healing, epithelial cancer, osteoarthritis, rheumatoid arthritis, cirrhosis and pulmonary fibrosis.

The FAP structure has been elucidated (PDB ID 1Z68) and is very similar to that of DPPIV. FAP is anchored to the plasma membrane by an uncleaved signal sequence of about 20 amino acids and has a short amino-terminal cytoplasmic domain of 6 amino acids. Most of the proteins containing the catalytic domain are exposed to the extracellular environment. The FAP glycoprotein is a homodimer consisting of two identical 97 kDa subunits. Each FAP monomer subunit consists of two domains, an αβ hydrolase domain (amino acids 27-53 and 493-760) and a β-propeller domain with 8 blades (amino acids 54-492), which encloses a large cavity It is out. A small pocket within this cavity at the interface of both domains contains the catalytic triad (Ser624, Asp702 and His734). FAP acquires its enzymatic activity upon subunit homodimerization, and besides its dipeptidyl peptidase activity, FAP also has type I collagen-specific gelatinase and endopeptidase activities. The β-propeller acts as a scaffold for protein-protein interactions and determines substrate and extracellular matrix (ECM) binding. In addition, β-propellers are involved in forming supramolecular complexes of FAP with other prolyl peptidases or other membrane-bound molecules. The formation of FAP and DPPIV heteromeric or tetrameric complexes was found to be associated with migrating cell infiltrates on the collagen matrix. Type I collagen induces a close association between FAP and β1 integrin, thereby playing a major organizational role in the formation and adhesion of invading processes. Although the mechanisms involved are not understood in detail, the formation of such proteinase-rich membrane domains in front of cell invasion contributes to directional pericellular ECM degradation. This may indicate that FAP and ECM interactions are closely related to invasive cell behavior by affecting cell adhesion, migration, proliferation and apoptosis via the integrin pathway, as well as in pathogenesis and progression It shows that it can support the role of FAP. In summary, FAP is recognized as a multifunctional protein that performs its biological functions in a cell-dependent manner through a combination of its protease activity and the ability to form complexes with other cell surface molecules. Overexpression of FAP in epithelial and fibroblast cell lines indicates a clinical situation where malignant behavior is promoted and cell expression levels of FAP correlate with worse clinical outcomes.

  Through the paracrine signaling molecule, cancer cells activate stromal fibroblasts and induce FAP expression, which in turn affects cancer cell proliferation, invasion and migration. Recent studies have demonstrated that TGF-β is a major factor in promoting FAP protein expression (Chen, H et al. (2009) Exp and Molecology, doi: 10.016 / j.yexmp. 2009). .09.001). FAP is highly expressed on reactive stromal fibroblasts in 90% of human epithelial cancers, including those of breast, lung, colorectal and ovary (Garin-Cesa, P et al. (1990) PNAS USA87. : 7236-7239). Chen et al. Recently showed that FAPα affects HO-8910PM ovarian cancer cell invasion, proliferation and migration (Chen, H et al. (2009) Exp and Molecology, doi: 10.016 / j. yexmp. 2009.9.0.001).

  FAP can be targeted by binding to the antigen and sterically blocking its interaction with biologically relevant molecules. Alternatively, or additionally, cross-linking a FAP molecule with another FAP molecule or a different molecule, such as an antigen on the surface of a cancer cell, can be achieved using multispecificity, such as a bispecific antibody molecule. This cross-linking increases the visibility of cells carrying the antigen to the immune system, which can then activate the immune system to neutralize or destroy it.

  Tumor associated macrophages (TAM) are thought to express TREM1, CD204, CD68 (alone or in combination with CD163 or CD206). These markers can be used to target TAM.

  Adenoviruses of the present disclosure have the ability to infect tumor cells and are specifically selected to preferentially infect tumor cells. Oncolytic viral infection causes the death and lysis of cancer cells with the release of newly generated viral particles. Integrated transgenes encoding antibodies, BiTE and other “payloads” are newly synthesized and actively secreted by tumor cells before they die, and some molecules are also released upon cell lysis Is done.

  Antibody molecules with a short half-life may be particularly suitable for use in the present disclosure as the body rapidly clears the molecule when available systemically, which minimizes off-target effects. is there.

  NKT cells have the ability to target and destroy tumor-associated macrophages. However, their activity appears to be inhibited by the hypoxic environment of the tumor. This activity can be restored, for example, by providing ILKT and / or IL-15 encoded by the virus of the present disclosure to NKT cells.

  Thus, in one embodiment, a virus according to the present disclosure further encodes an activating cytokine and NKT cells selected from, for example, IL-2, IL-15 and combinations thereof. The gene encoding the cytokine may be at the same position as or different from the gene encoding the antibody molecule selected independently from E1, E3, E4, BX and BY, for example.

  Thus, adenoviruses according to the present disclosure have at least two or three mechanisms for damaging the tumor, including indirect mechanisms that compromise the tumor stroma.

  As used herein, a transgene refers to a gene that has been inserted into a genomic sequence, which is a gene that is unnatural (exogenous) to the virus, or that is not normally found at that particular location in the virus. is there. Examples of transgenes are shown below. As used herein, a transgene also includes a functional fragment of a gene that, when inserted, is part of a gene that is suitable to perform the function or majority of the function of a full-length gene.

  Transgenes and coding sequences are used interchangeably herein in the context of insertion into the viral genome, unless otherwise noted. As used herein, a coding sequence refers to a DNA sequence that encodes, for example, a functional RNA, peptide, polypeptide or protein. Typically, the coding sequence is a transgene cDNA encoding a functional RNA, peptide, polypeptide or protein of interest. The functional RNAs, peptides, polypeptides and proteins of interest are described below.

  Clearly the viral genome contains the coding sequence of DNA. Endogenous (naturally occurring genes) in a viral genomic sequence are subject to the context of this specification unless they have been modified by recombinant techniques such that they are in a non-natural location or in a non-natural environment. Is not considered a transgene.

  In one embodiment, a transgene, as used herein, refers to a segment of DNA comprising a gene or cDNA sequence that has been isolated from one organism and introduced into a different organism, ie, a virus of the present disclosure. . In one embodiment, this non-natural segment of DNA may retain the ability to produce functional RNA, peptides, polypeptides or proteins.

  Thus, in one embodiment, the inserted transgene encodes a human or humanized protein, polypeptide or peptide.

  In one embodiment, the inserted transgene is a non-human protein, polypeptide or peptide (such as a non-human mammalian protein, polypeptide or peptide, etc.) derived from, for example, mouse, rat, rabbit, camel, llama or the like. ) Or an RNA molecule. Advantageously, the viruses of the present disclosure allow the transgene to be transported into cancerous cells. Thus, responses to non-human sequences (such as proteins) produced by human patients can be minimized by this intracellular delivery.

  A DNA sequence may contain two or more transgenes, for example 1, 2, 3 or 4 transgenes such as 1 or 2.

  A transgene cassette may contain two or more transgenes, for example 1, 2, 3 or 4 transgenes such as 1 or 2.

  In one or more embodiments, the cassettes are arranged as shown in one or more drawings or examples.

  As used herein, a transgene cassette refers to a DNA sequence that encodes one or more transgenes in the form of one or more coding sequences and one or more regulatory elements.

  The transgene cassette can encode one or more monocistronic and / or polycistronic mRNA sequences.

  In one embodiment, the transgene or transgene cassette encodes a monocistronic or polycistronic mRNA, for example, the cassette is located at a position under the control of an endogenous promoter or an exogenous promoter or combinations thereof. Suitable for insertion into.

  Monocistronic mRNA as used herein refers to an mRNA molecule that encodes a single functional RNA, peptide, polypeptide or protein.

  In one embodiment, the transgene cassette encodes a monocistronic mRNA.

  In one embodiment, a transgene cassette in the context of a cassette encoding a monocistronic mRNA is an exogenous promoter (which is a regulatory sequence that determines where and when the transgene is activated) or a splice site (where the mRNA molecule is A regulatory segment that determines when to be cleaved by the spliceosome), a DNA segment optionally comprising a coding sequence (ie, a transgene), usually derived from the cDNA of the protein of interest, and a poly A signal Means a DNA segment optionally comprising a sequence and a terminator sequence.

  In one embodiment, the transgene cassette may encode one or more polycistronic mRNA sequences.

  As used herein, polycistronic mRNA refers to an mRNA molecule that encodes two or more functional RNAs, peptides or proteins, or combinations thereof. In one embodiment, the transgene cassette encodes polycistronic mRNA.

  In one embodiment, the transgene cassette in the context of a cassette encoding polycistronic mRNA is an exogenous promoter (which is a regulatory sequence that determines where and when the transgene is active) or a splice site (when the mRNA molecule is A DNA segment that optionally includes two or more coding sequences (ie, transgenes), usually derived from the cDNA of the protein or peptide of interest, which is a regulatory sequence that determines whether it is cleaved by the spliceosome Thus, for example, each coding sequence includes DNA segments that are separated by either an IRES or 2A peptide. Following the last coding sequence to be transcribed, the cassette may optionally include a poly A sequence and a terminator sequence.

  In one embodiment, the transgene cassette encodes a monocistronic mRNA followed by a polycistronic mRNA. In another embodiment, the transgene cassette encodes polycistronic mRNA followed by monocistronic mRNA.

In one embodiment, the adenovirus is a human adenovirus. As used herein, “adenovirus”, “serotype” or “adenovirus serotype” is classified into subgroups AF and has not yet been identified or even further unclassified. Refers to any adenovirus that can belong to some of the more than 50 currently known adenovirus serotypes. For example, as shown in Table 1, Strauss, “Adenovirus effects in humans” in The Adenoviruses, Ginsberg, ea. , Plenum Press, New York, NY, pp. 451-596 (1984) and Shenk, '' Adnoviridae: The Viruses and Their Replication '' in Fields Virology, Vol. 2, Fourth Edition, Knipe, 35ea. Lippincott Williams & Wilkins, pp. 2265-2267 (2001).
Table 1

  Adenoviruses of the present disclosure are subgroup B viruses, ie, Ad11, particularly Ad11p (Slobitski strain), and derivatives thereof, such as EnAd.

  Adenoviruses are assigned to their group / serotype based on capsids such as hexons and / or fibers.

  Adenoviruses of the present disclosure are not A, C, D, E or F group viruses. The viruses of the present disclosure do not contain adenovirus death protein.

  In one embodiment, the adenovirus of the present disclosure is a chimera. If the adenovirus is chimeric, the outer capsid features will be used to determine the serotype. As used herein, a chimera refers to a virus comprising DNA from at least two different viral serotypes including different serotypes within the same group.

  In one embodiment, the oncolytic virus is derived from the same serotype, eg, Ad11, in particular Ad11p, eg, fiber, hexon and penton found at positions 30812-31789, 18254-21100 and 13682-15367 of the latter genomic sequence. It has a protein and in the latter genomic sequence the nucleotide position is relative to Genbank ID 21307399 (registration number: GC689208).

  In one embodiment, the adenovirus is enadenotucirev (also known as EnAd and formerly EnAd). As used herein, enadenochucilv refers to the chimeric adenovirus of SEQ ID NO: 38. It is a replication competent oncolytic chimeric adenovirus with enhanced therapeutic properties compared to wild type adenovirus (see WO 2005/118825). EnAd has a DNA derived from Ad11p and Ad3 and a chimeric E2B region characterized by a deletion in E3 / E4. The structural change of enadenoticirev results in a genome that is about 3.5 kb smaller than Ad11p, thereby providing additional “space” for transgene insertion.

  Enadenotucirev (EnAd) is a chimeric oncolytic adenovirus, formerly known as EnAd (WO2005 / 118825), with fibers, pentons and hexons from Ad11p, so it is subgroup B It is a virus. It has a chimeric E2B region containing DNA from Ad11p and Ad3. In EnAd, almost all of the E3 area and a part of the E4 area are deleted. Therefore, it has significant space in the genome to accommodate additional genetic material while remaining viable. Furthermore, since EnAd is a subgroup B adenovirus, existing immunity in humans is less common than, for example, Ad5. Other examples of chimeric oncolytic viruses with Ad11 fiber, penton and hexon include OvAd1 and OvAd2 (see WO 2006/060314).

  EnAd preferentially infects tumor cells and appears to replicate rapidly within these cells, causing cell lysis. This, in turn, can generate an inflammatory immune response, thereby stimulating the body to fight cancer. Part of EnAd's success has been postulated to be related to rapid replication of the virus in vivo.

  EnAd selectively lyses tumor cells, but is a transgene such as a transgene that enhances the therapeutic activity of a virus or encodes a cell signaling protein or antibody, or a transgene that encodes an entity that stimulates a cell signaling protein. Arming it can introduce additional beneficial properties such as reducing the side effects of viruses.

  Advantageously arming a virus with DNA encoding a specific protein such as BiTE that can be expressed in cancer cells, eg, by making the cell more visible to the immune system or targeting a therapeutic gene / protein to a tumor By preferentially delivering to cells, it may be possible to more effectively fight tumor cells using the body's own defenses.

  In addition, the ability to insert a reporter transgene into the genome can be useful for clinical or preclinical studies.

  It is important that the expression of the transgene does not adversely affect viral replication or other advantageous properties. Thus, the gene or genes must be inserted in a location that does not impair replication ability and other advantageous properties of the virus. In addition, because the adenovirus genome is dense, it may be difficult to find a suitable location for inserting the transgene. This also limits the size of the transgene that can be accommodated.

  OvAd1 and OvAd2 are also chimeric adenoviruses similar to enadenotucirev, and they also have additional “space” in the genome (see WO2008 / 080003). Thus, in one embodiment, the adenovirus is OvAd1 or OvAd2.

  In one embodiment, the adenovirus is oncolytic. As used herein, an oncolytic adenovirus refers to an adenovirus that preferentially kills cancer cells compared to non-cancerous cells.

  In one embodiment, the oncolytic virus is apoptotic. That is, it promotes programmed cell death.

  In one embodiment, the oncolytic virus is cytolytic. The cytolytic activity of the disclosed oncolytic adenoviruses can be determined in representative tumor cell lines and adenoviruses belonging to subgroup C such as Ad5 are used as standards (ie, potency of 1). Data can be converted to efficacy measures. A suitable method for determining cytolytic activity is the MTS assay (see Example 4 of WO 2005/118825, FIG. 2, which is incorporated herein by reference).

  In one embodiment, the oncolytic virus is necrotic. That is, it causes or accelerates cell necrosis or immunogenic cell death. In one embodiment, necrotic cell death is advantageous because it induces and induces a patient (host) immune response.

  Unless stated otherwise, adenovirus as used herein also refers to a replicable virus (eg, a replicative virus) and a replication-defective viral vector.

  As used herein, replicable refers to a virus that is capable of replication, or whose replication depends on factors within the cancer cell, such as upregulatory factors such as p53.

  In one embodiment, the virus is capable of replication. Replication capable in the context of the present specification refers to a virus that possesses all the mechanisms necessary for replication in cells in vitro and in vivo, ie without the aid of a packaging cell line. Viral vectors that can replicate in a complementary packaging cell line, eg, deleted in the E1 region, are not replication competent viruses in this context.

  Viral vectors are replication deficient and require packaging cells to provide a complementary gene to allow replication.

  As used herein, an adenoviral genome refers to a DNA sequence that encodes structural proteins and elements related to the function / life cycle of an adenovirus.

  All human adenoviral genomes examined to date have the same general organization, i.e. genes encoding specific functions are located at the same position in the viral genome (referred to herein as structural elements). Each end of the viral genome has a short sequence known as the terminal inversion sequence (or ITR) required for viral replication. The viral genome produces five families of five early transcription units (E1A, E1B, E2, E3, and E4), three delayed early units (IX, IVa2, and E2 late), and late mRNAs (L1-L5) One late unit (major late) to be processed. The proteins encoded by the early genes are primarily involved in the replication and regulation of the host cell response to infection, whereas the late genes encode viral structural proteins. Early genes are preceded by the letter E, and late genes are preceded by the letter L.

  Because the adenovirus genome is dense, ie, has few non-coding sequences, it can be difficult to find a suitable location for inserting the transgene. The inventors have identified two DNA regions where transgenes are permissible, and the identified sites are particularly suitable for accommodating complex transgenes such as those encoding antibodies. That is, the transgene is expressed without adversely affecting natural properties such as viral viability, oncolytic properties, or replication.

  In one embodiment, an oncolytic or partial oncolytic virus according to the present disclosure comprises a deletion of the E4 and / or E3 region, such as a partial deletion of the E4 region, or a complete deletion of the E3 region, or As exemplified by the sequences disclosed in the specification, it may be the result of a deletion of a part of the E4 region (such as E4orf4) and a complete deletion of the E3 region.

  In one embodiment, the oncolytic virus of the present disclosure is chimeric. As used herein, a chimera refers to a virus that contains DNA from two or more different serotypes and has oncolytic viral properties.

  In one embodiment, the oncolytic virus is EnAd or an active derivative thereof that retains the essential beneficial properties of the virus. EnAd is disclosed in WO 2005/118825 (incorporated herein by reference) and the entire viral sequence is provided herein as SEQ ID NO: 38. The chimeric E2B region is disclosed herein as SEQ ID NO: 71.

  Alternative oncolytic viruses include OvAd1 and OvAd2, which are disclosed as SEQ ID NOs: 2 and 3, respectively, in WO2008 / 080003 and are incorporated herein by reference.

  Advantageously, adenoviruses of the present disclosure exhibit similar viral activity, eg, replication and / or infectivity, profiles as EnAd after infection of a variety of different colon cancer cell lines in vitro.

Adenoviral Structural Elements The present disclosure also relates to novel sequences of viruses or viral components / constructs such as plasmids disclosed herein.

In one embodiment, the adenovirus has the formula (I):
5'ITR-B1-BA-B2-BX-BB-BY-B3-3'ITR (I)
Wherein B1 includes E1A, E1B or E1A-E1B, BA includes E2B-L1-L2-L3-E2A-L4, B2 is a bond or includes E3, and BX is a bond or a restriction A DNA sequence comprising a site, one or more transgenes (in particular a transgene encoding at least one BiTE according to the present disclosure under the control of an exogenous promoter) or both, wherein BB comprises L5, BY is a binding or restriction site, at least one transgene (especially under the control of an endogenous promoter such as MPL or under the control of an exogenous promoter such as the CMV promoter) A transgene encoding BiTE) or both, wherein B3 is a bond or contains E4, where BX and At least one of BY is not binding but contains a transgene or restriction site or both, and encodes a multispecific antigen molecule comprising at least two binding domains, wherein at least one of said domains is a surface on a T cell of interest A genome comprising a sequence of formula (I) that is specific for an antigen is included. In one embodiment, the adenovirus comprises a genome comprising a sequence of formula (I) to which B1 BX is bound.

  In one embodiment, the adenovirus comprises a genome comprising a sequence of formula (I), wherein BY is a bond.

In one embodiment, the adenovirus has the formula (Ia):
5′ITR-BA-B2-BX-BB-BY-B3-3′ITR (Ia)
Where BA includes E2B-L1-L2-L3-E2A-L4, B2 is a bond or includes E3, BX is a bond or a restriction site, one or more transgenes (in particular, A DNA sequence comprising, for example, a transgene encoding at least one BiTE according to the present disclosure under the control of an exogenous promoter) or both, BB comprises L5 and BY is a binding or restriction site, one Or a plurality of transgenes (especially a transgene encoding at least one BiTE according to the present disclosure under the control of an endogenous promoter such as MPL or under the control of an exogenous promoter such as the CMV promoter) or both A DNA sequence comprising: B3 is a bond or comprises E4, wherein at least one of BX and BY is not a bond and is at least Also one containing a transgene or restriction site, such as a transgene, comprising a genome comprising a sequence of formula (Ia).

  In one embodiment, the adenovirus comprises a genome comprising a sequence of formula (Ia), wherein BX is a bond.

  In one embodiment, the adenovirus comprises a genome comprising a sequence of formula (Ia), wherein BY is a bond.

In one embodiment, the adenovirus has the formula (Ib):
5′ITR-BA-BX-BB-BY-B3-3′ITR Ib (Ib)
Wherein BA comprises E2B-L1-L2-L3-E2A-L4 and BX is a binding or restriction site, one or more transgenes (especially for example under the control of an exogenous promoter At least one BiTE-encoding transgene) or both, wherein BB contains L5 and BY is a binding or restriction site, one or more transgenes (especially for example in MPL) A transgene encoding at least one BiTE according to the present disclosure, or both, under the control of an endogenous promoter such as or under the control of an exogenous promoter such as the CMV promoter, or is B3 binding? Or E4, wherein at least one of BX and BY is not a bond, but a transgene such as a transgene or a restriction site or Includes both, it comprises a genome comprising a sequence of formula (Ib).

  In one embodiment, the adenovirus comprises a genome comprising a sequence of formula (Ib), wherein BX is a bond.

  In one embodiment, the adenovirus comprises a genome comprising a sequence of formula (Ib), wherein BY is a bond.

In one embodiment, the adenovirus has the formula (Ic):
5′ITR-BA-B2-BX-BB-BY-3′ITR (Ic)
Where BA includes E2B-L1-L2-L3-E2A-L4, B2 is E3, BX is a binding or restriction site, one or more transgenes (especially for example of an exogenous promoter) A transgene encoding at least one BiTE according to the present disclosure under control) or both, wherein BB comprises L5 and BY is a binding or restriction site, one or more transgenes (In particular a transgene encoding at least one BiTE according to the present disclosure under the control of an endogenous promoter such as MPL or under the control of an exogenous promoter such as the CMV promoter) or both, Here, at least one of BX and BY is not a bond, but includes a transgene such as a transgene and / or a restriction site, Including the genome that contains an array of (Ic).

  In one embodiment, the adenovirus comprises a genome comprising a sequence of formula (Ic), wherein BX is a bond.

  In one embodiment, the adenovirus comprises a genome comprising a sequence of formula (Ic), wherein BY is a bond.

In one embodiment, the adenovirus has the formula (Id):
5′ITR-B1-BA-BX-BB-BY-B3-3′ITR (Id)
Where B1 includes E1A, E1B, or E1A-E1B, BA includes E2B-L1-L2-L3-E2A-L4, and BX is a binding or restriction site, one or more transgenes ( In particular, a DNA sequence comprising, for example, a transgene encoding at least one BiTE according to the present disclosure under the control of an exogenous promoter) or both, BB comprises L5 and BY is a binding or restriction site, One or more transgenes (in particular a transgene encoding at least one BiTE according to the present disclosure under the control of an endogenous promoter such as MPL or under the control of an exogenous promoter such as the CMV promoter) or both B3 is a bond or includes E4, wherein at least one of BX and BY is not a bond Ku, the transgene comprises restriction sites, or both, comprises a genome comprising a sequence of formula (Id).

  In one embodiment, the adenovirus comprises a genome comprising a sequence of formula (Id) to which BX is bound.

  In one embodiment, the adenovirus comprises a genome comprising a sequence of formula (Id) where BY is a bond.

In one embodiment, the adenovirus has the formula (Ie):
5′ITR-B1-BA-B2-BB-BY-3′ITR (Ie)
Where B1 includes E1A, E1B, or E1A-E1B, BA includes E2B-L1-L2-L3-E2A-L4, B2 includes E3, BB includes L5, and BY is a bond Or a DNA sequence comprising one or more transgenes encoding at least one BiTE according to the present disclosure (for example under the control of an endogenous promoter such as MPL or under the control of an exogenous promoter such as the CMV promoter) Wherein at least one of BX and BY is not a bond but comprises a genome comprising a sequence of formula (Ie) comprising a transgene, a restriction site, or both.

  In one embodiment, formulas (I), (Ia), (Ib) comprising a transgene, a restriction site, or both, wherein BX and BY are not binding and both BX and BY are transgenes. , (Ic), (Id) or (Ie).

  In one embodiment of formulas (I), (Ia), (Ib), (Ic) or (Id), only BX codes one or two BiTEs, for example one BiTE (BY does not code BiTE) In particular, the one or more BiTEs are under the control of an exogenous promoter such as the CMV promoter. In one embodiment of Formula (I), (Ia), (Ib), (Ic), or (Id), only BY encodes one or two BiTEs, eg, one BiTE (BX does not encode BiTE) In particular, the one or more BiTEs are under the control of an endogenous promoter such as MPL, or under the control of an exogenous promoter such as the CMV promoter. In one embodiment of formula (I), (Ia), (Ib), (Ic) or (Id), BX encodes BiTE (eg, under the control of an exogenous promoter such as the CMV promoter) and BY is Encodes BiTE (eg, under the control of an endogenous promoter such as MPL or under the control of an exogenous promoter such as the CMV promoter).

  Binding refers to a covalent bond that connects one DNA sequence to another DNA sequence, eg, connecting one part of the viral genome to another part. Thus, when a variable in formula (I), (Ia), (Ib), (Ic), (Id) or (Ie) herein represents a bond, there is a feature or element represented by that bond No, ie deleted.

  Since the structures of adenoviruses are generally similar, the following elements are discussed with respect to structural elements known to those skilled in the art and commonly used terms that refer to them. When an element is referred to herein, we refer to a DNA sequence that encodes that element or a DNA sequence that encodes the same structural protein of the element in an adenovirus. The latter is appropriate because of the redundancy of the DNA code. In order to obtain optimized results, it is necessary to consider the virus preference for codon usage.

  Any structural element derived from an adenovirus used in the virus of the present disclosure comprises or consists of a native sequence or is at least 95% over a given length, eg, 96%, 97%, 98%, 99 % Or 100% similarity. The original sequence can be modified to omit 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the genetic material. Those skilled in the art recognize that the viral reading frame must not be destroyed when changes are made so that expression of the structural protein is destroyed.

  In one embodiment, the given element is a full length sequence, ie a full length gene.

  In one embodiment, a given element is less than full length and retains the same or corresponding function as the full length sequence.

  In one embodiment for a given element that is optional in the constructs of the present disclosure, the DNA sequence may be less than full length and not functional.

  Structural genes encoding adenovirus structural or functional proteins are generally linked by non-coding regions of DNA. Thus, there is some flexibility in where to “cut” the genomic sequence of the structural element of interest (particularly its non-coding region) for the purpose of inserting a transgene into the virus of the present disclosure. Thus, for the purposes of this specification, that element will be considered a reference structural element unless it is adapted for the purpose and does not encode extraneous material. Thus, if appropriate, a gene will be associated with a suitable non-coding region, for example as found in the native structure of a virus.

  Thus, in one embodiment, inserts such as DNA encoding restriction sites and / or transgenes are inserted into non-coding regions of genomic viral DNA, such as introns or intergenic sequences. This several non-coding regions of adenovirus have been described as potentially having functions in, for example, alternative splicing, transcriptional or translational regulation and may need to be taken into account.

  Sites identified herein that relate to the L5 region (eg, between the L5 and E4 regions) are complex entities such as RNAi, cytokines, single chains, or multimeric proteins such as antibodies such as BiTE. Suitable to accommodate various DNA sequences encoding.

  As used herein, a gene refers to coding and any non-coding sequences associated with it, such as introns and related exons. In one embodiment, a gene comprises or consists of only essential structural components, eg, coding regions.

  The following follows a discussion of specific structural elements of adenovirus.

  Terminal inversion sequences (ITRs) are common to all known adenoviruses, so named because of their symmetry, and are the origin of viral chromosomal replication. Another property of these sequences is their ability to form hairpin structures.

  As used herein, 5 'ITR refers to part or all of an ITR from the 5' end of an adenovirus that retains the function of the ITR when incorporated at the appropriate location in the adenovirus. In one embodiment, the 5 ′ ITR is a sequence from about 1 bp to 138 bp of SEQ ID NO: 38, or a sequence 90, 95, 96, 97, 98 or 99% identical to it along the entire length, in particular about 1 bp of SEQ ID NO: 38. It comprises or consists of a sequence consisting of ˜138 bp.

  As used herein, 3 'ITR refers to part or all of an ITR from the 3' end of an adenovirus that retains the function of the ITR when incorporated at the appropriate location in the adenovirus. In one embodiment, the 3 ′ ITR is about 32189 bp to 32326 bp of SEQ ID NO: 38, or a sequence 90, 95, 96, 97, 98 or 99% identical to it along the entire length, in particular about 32189 bp of SEQ ID NO: 38. It comprises or consists of a sequence consisting of ~ 32326 bp.

  B1 as used herein encodes part or all of adenovirus-derived E1A, part or all of adenovirus E1B region, and independently part or all of adenovirus E1A and E1B regions Refers to the DNA sequence.

  If B1 is binding, the E1A and E1B sequences will be excluded from the virus. In one embodiment, B1 is a bond and thus the virus is a vector.

  In one embodiment, B1 further comprises a transgene. The E1 region can be inserted into the E1 region in a destructive manner (ie, “in the middle” of the sequence) or can contain a transgene, or part or all of the E1 region can contain genetic material It is known in the art that deletions can be made to provide more space for.

  As used herein, E1A refers to a DNA sequence that encodes part or all of the adenovirus E1A region. The latter here refers to the polypeptide / protein E1A. Functions such as that the protein encoded by the E1A gene has the same function as the wild type (ie, the corresponding non-mutated protein), improved function compared to the wild type protein, and no function compared to the wild type protein It may be mutated to have conservative or non-conservative amino acid changes, such as reduced, or have a new function compared to wild-type protein or optionally a combination thereof.

  As used herein, E1B refers to a DNA sequence that encodes part or all of an adenovirus E1B region (ie, a polypeptide or protein), and the protein encoded by the E1B gene / region is, for example, the same as the wild type Functional (ie corresponding non-mutated protein), improved function compared to wild-type protein, reduced function such as lack of function compared to wild-type protein, or wild-type protein or combinations thereof as required It may be mutated to have conservative or non-conservative amino acid changes, such as having a new function compared to.

  Thus, B1 may or may not be modified relative to a wild type E1 region such as wild type E1A and / or E1B. One skilled in the art can readily identify whether E1A and / or E1B is present, or (partly) deleted or mutated.

  Wild type as used herein refers to a known adenovirus. Known adenoviruses have been identified and named regardless of whether sequences are available.

  In one embodiment, B1 has the sequence of 139 bp to 3932 bp of SEQ ID NO: 38.

  BA, as used herein, refers to a DNA sequence that encodes the E2B-L1-L2-L3-E2A-L4 region, including any non-coding sequence, as appropriate. Generally this sequence will not contain the transgene. In one embodiment, the sequence is a known adenovirus, such as the serotypes shown in Table 1, in particular group B viruses, such as Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad51, or combinations thereof, For example, it is substantially similar or identical to a contiguous sequence from Ad3, Ad11 or combinations thereof. In one embodiment, E2B-L1-L2-L3-E2A-L4 is meant to include these elements and other structural elements related to that region, for example BA generally includes, for example: IV2A IV2a- Includes sequences encoding protein IV2a, such as E2B-L1-L2-L3-E2A-L4.

  In one embodiment, the E2B region is chimeric. That is, it contains DNA sequences from two or more different adenovirus serotypes, eg, Ad3 and Ad11, eg, Ad11p. In one embodiment, the E2B region has a sequence of 5068 bp to 10355 bp of SEQ ID NO: 38, or a sequence that is 95%, 96%, 97%, 98% or 99% identical thereto.

  In one embodiment, E2B in component BA comprises the sequence shown in SEQ ID NO: 71 (corresponding to SEQ ID NO: 3 disclosed in WO2005 / 118825).

  In one embodiment, the BA has the sequence of 3933 bp to 27184 bp of SEQ ID NO: 38.

  As used herein, E3 refers to a DNA sequence (ie, protein / polypeptide) that encodes part or all of the adenovirus E3 region, and the protein encoded by the E3 gene is, for example, the same as the wild type Functional (corresponding unmutated protein), improved function compared to wild type protein, reduced function such as no function compared to wild type protein, or wild type protein or combinations thereof as required It may be mutated to have conservative or non-conservative amino acid changes, such as having a new function in comparison.

  In one embodiment, the E3 region is an adenovirus serotype as shown in Table 1, or a combination thereof, in particular a group B serotype such as Ad3, Ad7, Ad11 (especially Ad11p), Ad14, Ad16, Ad21, Ad34, Ad35, Ad51. Or a combination thereof, for example, Ad3, Ad11 (particularly Ad11p) or a combination thereof.

  In one embodiment, the E3 region is partially deleted, for example 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45% , 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% are deleted.

  In one embodiment, B2 is a bond and there is no DNA encoding the E3 region.

  In one embodiment, the DNA encoding the E3 region can be replaced or interrupted by the transgene. As used herein, “E3 region replaced by a transgene as used herein includes that part or all of the E3 region is replaced by a transgene.

  In one embodiment, the B2 region comprises the sequence from 27185 bp to 28165 bp of SEQ ID NO: 38.

  In one embodiment, B2 consists of the sequence from 27185 bp to 28165 bp of SEQ ID NO: 38.

  BX as used herein refers to a DNA sequence near the 5 'end of the L5 gene in BB. As used herein, near or proximal to the 5 ′ end of the L5 gene is a non-coding region adjacent to (or adjacent to) or essentially related to the 5 ′ end of the L5 gene, ie, the L5 gene. Refers to a non-coding region that is adjacent to or adjacent to the extreme end of or essentially associated therewith. Alternatively, near or proximal to the L5 gene may refer to being close to the L5 gene such that there is no coding sequence between the BX region and the 5 'end of the L5 gene.

  Thus, in one embodiment, BX is directly linked to the base of L5, eg representing the start of the coding sequence of the L5 gene.

  Thus, in one embodiment, BX binds directly to the base of L5, eg, representing the beginning of a non-coding sequence, or directly to a non-coding region naturally associated with L5. As used herein, naturally associated non-coding region L5 refers to a portion of the L5 gene or a portion of all non-coding regions that are contiguous but not part of other genes.

  In one embodiment, BX comprises the sequence of SEQ ID NO: 39. This sequence is an artificial non-coding sequence, for example, a DNA sequence containing a transgene (or transgene cassette), a restriction site or a combination thereof may be inserted therein. This sequence acts as a buffer in that it can minimize the disruptive impact on virus stability and viability while allowing some flexibility to the exact location of the transgene. This is advantageous.

  Insertion is from the 5 'end in SEQ ID NO: 39 to the 3' end, or any point between bp 1-201, eg base pairs 1/2, 2/3, 3/4, 4/5, 5/6. 6/7, 7/8, 8/9, 9/10, 10/11, 11/12, 12/13, 13/14, 14/15, 15/16, 16/17, 17/18, 18 / 19, 19/20, 20/21, 21/22, 22/23, 23/24, 24/25, 25/26, 26/27, 27/28, 28/29, 29/30, 30/31 31/32, 32/33, 33/34, 34/35, 35/36, 36/37, 37/38, 38/39, 39/40, 40/41, 41/42, 42/43, 43 / 44, 44/45, 45/46, 46/47, 47/48, 48/49, 49/50, 50/51, 51 / 2, 52/53, 53/54, 54/55, 55/56, 56/57, 57/58, 58/59, 59/60, 60/61, 61/62, 62/63, 63/64, 64/65, 65/66, 66/67, 67/68, 68/69, 69/70, 70/71, 71/72, 72/73, 73/74, 74/75, 75/76, 76 / 77, 77/78, 78/79, 79/80, 80/81, 81/82, 82/83, 83/84, 84/85, 85/86, 86/87, 87/88, 88/89, 89/90, 90/91, 91/92, 92/93, 93/94, 94/95, 95/96, 96/97, 97/98, 98/99, 99/100, 100/101, 101 / 102, 102/103, 103/104, 104/105, 1 5/106, 106/107, 107/108, 108/109, 109/110, 110/111, 111/112, 112/113, 113/114, 114/115, 115/116, 116/117, 117 / 118, 118/119, 119/120, 120/121, 121/122, 122/123, 123/124, 124/125, 125/126, 126/127, 127/128, 128/129, 129/130, 130/131, 131/132, 132/133, 133/134, 134/135, 135/136, 136/137, 137/138, 138/139, 139/140, 140/141, 141/142, 142 / 143, 143/144, 144/145, 145/146, 146/147 147/148, 148/149, 150/151, 151/152, 152/153, 153/154, 154/155, 155/156, 156/157, 157/158, 158/159, 159/160, 160 / 161, 161/162, 162/163, 163/164, 164/165, 165/166, 166/167, 167/168, 168/169, 169/170, 170/171, 171/172, 172/174 174/175, 175/176, 176/177, 177/178, 178/179, 179/180, 180/181, 181/182, 182/183, 183/184, 184/185, 185/186, 186 / 187, 187/188, 189/190, 190/191, 191 / 92,192 / 193, 193/194, 194/195, 195/196, 196 / 197,197 / 198,198 / 199,199 / 200, or can occur anywhere between 200/201.

  In one embodiment, BX comprises SEQ ID NO: 39 and the DNA sequence is inserted between base pair 27 and base pair 28 or at a position corresponding to 28192 bp to 28193 bp of SEQ ID NO: 38.

  In one embodiment, the insert is a restriction site insert. In one embodiment, the restriction site insert comprises one or two restriction sites. In one embodiment, the restriction site is a 19 bp restriction site insert comprising two restriction sites. In one embodiment, the restriction site insert is a 9 bp restriction site insert comprising one restriction site. In one embodiment, the restriction site insert comprises one or two restriction sites and at least one transgene, such as one or two transgenes. In one embodiment, the restriction site is a 19 bp restriction site insert comprising two restriction sites and at least one transgene, such as one or two transgenes. In one embodiment, the restriction site insert is a 9 bp restriction site insert comprising one restriction site and at least one transgene, for example one, two or three transgenes, such as one or two. In one embodiment, two restriction sites sandwich one or more, eg, two transgenes (eg, within a transgene cassette). In one embodiment, when the BX contains two restriction sites, the restriction sites are different from each other. In one embodiment, the one or more restriction sites in BX are not naturally present in the particular adenoviral genome into which they are inserted. In one embodiment, the one or more restriction sites in BX are different from other restriction sites located elsewhere in the adenovirus genome, eg, introduced into naturally occurring restriction sites and / or BY. It is different from restriction sites introduced into other parts of the genome, such as restriction sites that have been introduced. Thus, in one embodiment, one or more restriction sites can specifically cleave that portion of DNA.

  Advantageously, the use of “unique” restriction sites provides selectivity and control when the viral genome is simply cleaved with the appropriate restriction enzymes.

  As used herein, specifically cleaving means that the use of an enzyme specific for a restriction site usually causes the virus to be in one position, but sometimes only in two desired positions. Refers to cutting. As used herein, a pair of positions refers to two adjacent restriction sites that are designed to be cleaved by the same enzyme (ie, cannot be distinguished from each other).

  In one embodiment, the restriction site insert is SEQ ID NO: 50.

  In one embodiment, BX has the sequence of 28166 bp to 28366 bp of SEQ ID NO: 38.

  In one embodiment, BX is a bond.

  In one embodiment, the BX contains restriction sites, eg 1, 2, 3 or 4 restriction sites such as 1 or 2. In one embodiment, BX comprises at least one transgene, for example one or two transgenes. In one embodiment, BX is at least one transgene, eg 1 if the restriction site sandwiches a DNA sequence comprising the gene or genes that allow them to be specifically excised and / or replaced from the genome. Or two transgenes and one or more restriction sites, eg 2 or 3 restriction sites. Or, for example, if there are two transgenes, restriction sites can sandwich each gene, and three different restriction sites are required to ensure that the gene is selectively excised and / or replaced. The In one embodiment, one or more, eg, all transgenes are in the form of a transgene cassette. In one embodiment, BX comprises SEQ ID NO: 39. In one embodiment, SEQ ID NO: 39 is interrupted by, for example, a transgene. In an embodiment, SEQ ID NO: 39 is not interrupted. In one embodiment, BX does not include a restriction site. In one embodiment, BX is a bond. In one embodiment, Bx comprises or consists of one or more transgenes.

  In one embodiment, BY comprises restriction sites, eg 1, 2, 3 or 4 restriction sites such as 1 or 2. In one embodiment, BY comprises at least one transgene, for example one or two transgenes. In one embodiment, BY is at least one transgene, eg 1 if the restriction site sandwiches a DNA sequence comprising genes or genes that allow them to be specifically excised and / or replaced from the genome. Or two transgenes and one or more restriction sites, eg 2 or 3 restriction sites. Or, for example, if there are two transgenes, restriction sites can sandwich each gene, and three different restriction sites are required to ensure that the gene is selectively excised and / or replaced. The In one embodiment, one or more, eg, all transgenes are in the form of a transgene cassette. In one embodiment, BY comprises SEQ ID NO: 40. In one embodiment, SEQ ID NO: 40 is interrupted by, for example, a transgene. In an embodiment, SEQ ID NO: 40 is not interrupted. In one embodiment, BY does not include a restriction site. In one embodiment, BY is a bond. In one embodiment, BY comprises or consists of one or more transgenes.

  In one embodiment, BX and BY each contain restriction sites, eg 1, 2, 3 or 4 restriction sites such as 1 or 2. In one embodiment, BX and BY each comprise at least one transgene, for example one or two transgenes. In one embodiment, BX and BY are each at least one transgene, particularly when the restriction site sandwiches a gene or DNA sequence comprising a gene that allows it to be specifically excised and / or replaced from the genome. For example, one or two transgenes and one or more restriction sites, for example two or three restriction sites. Or, for example, if there are two transgenes, restriction sites can sandwich each gene, and three different restriction sites are required to ensure that the gene is selectively excised and / or replaced. The In one embodiment, one or more, eg, all transgenes are in the form of a transgene cassette. In one embodiment, BX and BY comprise SEQ ID NO: 39 and SEQ ID NO: 40, respectively. In one embodiment, BX and BY do not contain restriction sites. In one embodiment, BX is a bond and BY is not a bond. In one embodiment, BY is a bond and BX is not a bond.

  BB as used herein refers to a DNA sequence that encodes the L5 region. As used herein, the L5 region refers to a DNA sequence comprising a gene encoding a fiber polypeptide / protein, as appropriate in context. The fiber gene / region encodes a fiber protein that is the major capsid component of adenovirus. The fiber functions in receptor recognition and contributes to the ability of adenovirus to selectively bind and infect cells.

  In the viruses of the present disclosure, the fiber can be from any serotype 11 adenovirus strain, such as Ad11p.

  In one embodiment, the BB has a sequence of 28367 bp to 29344 bp of SEQ ID NO: 38.

  As used herein, the DNA sequence for BY refers to the DNA sequence near the 3 'end of the BB L5 gene. As used herein, near or near the 3 ′ end of the L5 gene is adjacent (proximal) to or essentially related to the 3 ′ end of the L5 gene, ie, the L5 gene Refers to a non-coding region adjacent to, or essentially related to, the 3 ′ extreme end (ie, all or part of a non-coding sequence endogenous to L5). Alternatively, near or proximal to the L5 gene may refer to being close to the L5 gene such that there is no coding sequence between the BY region and the 3 'end of the L5 gene.

  Thus, in one embodiment, BY binds directly to the base of L5 representing the “end” of the coding sequence.

  Thus, in one embodiment, BY binds directly to the base of L5 representing the “end” of the non-coding sequence, or directly to a non-coding region naturally associated with L5.

  As used herein, they are used interchangeably in nature and nature. In one embodiment, BY comprises the sequence of SEQ ID NO: 40. This sequence is a non-coding sequence, and for example, a DNA sequence containing a transgene (or transgene cassette), a restriction site or a combination thereof may be inserted. This sequence is like a buffer in that it can minimize the disruptive impact on virus stability and viability while allowing some flexibility to the exact location of the transgene. This is advantageous because it works.

  Insertion is from the 5 ′ end to the 3 ′ end in SEQ ID NO: 40, or any point between bp 1-35, eg, base pairs 1/2, 2/3, 3/4, 4/5, 5/6. 6/7, 7/8, 8/9, 9/10, 10/11, 11/12, 12/13, 13/14, 14/15, 15/16, 16/17, 17/18, 18 / 19, 19/20, 20/21, 21/22, 22/23, 23/24, 24/25, 25/26, 26/27, 27/28, 28/29, 29/30, 30/31 , 31/32, 32/33, 33/34, or 34/35.

  In one embodiment, BY comprises SEQ ID NO: 40, and the DNA sequence is inserted between base pair 12 and base pair 13 or at a position corresponding to 29356 bp to 29357 bp of SEQ ID NO: 38. In one embodiment, the insert is a restriction site insert. In one embodiment, the restriction site insert comprises one or two restriction sites. In one embodiment, the restriction site is a 19 bp restriction site insert comprising two restriction sites. In one embodiment, the restriction site insert is a 9 bp restriction site insert comprising one restriction site. In one embodiment, the restriction site insert comprises one or two restriction sites and at least one transgene, such as one or two or three transgenes, such as one or two transgenes. In one embodiment, the restriction site is a 19 bp restriction site insert comprising two restriction sites and at least one transgene, such as one or two transgenes. In one embodiment, the restriction site insert is a 9 bp restriction site insert comprising one restriction site and at least one transgene, eg, one or two transgenes. In one embodiment, two restriction sites sandwich one or more, eg, two transgenes (eg, within a transgene cassette). In one embodiment, when BY contains two restriction sites, the restriction sites are different from each other. In one embodiment, the one or more restriction sites in BY are not naturally occurring (eg, unique) in the particular adenoviral genome into which they are inserted. In one embodiment, the one or more restriction sites in the BY are different from other restriction sites located elsewhere in the adenovirus genome, eg, a naturally occurring restriction site or of a genome such as BX. It is different from the restriction sites introduced in other parts. Thus, in one embodiment, one or more restriction sites can specifically cleave that portion of DNA.

  In one embodiment, the restriction site insert is SEQ ID NO: 51.

  In one embodiment, BY has the sequence of 29345 bp to 29379 bp of SEQ ID NO: 38.

  In one embodiment, BY is a bond.

  In one embodiment, the insert is after bp 12 of SEQ ID NO: 40.

  In one embodiment, the insert is approximately 29356 bp of SEQ ID NO: 38.

  In one embodiment, the insert is a transgene cassette comprising one or more transgenes, eg 1, 2 or 3 transgenes such as 1 or 2.

  As used herein, E4 refers to a DNA sequence (ie, a polypeptide / protein region) that encodes part or all of an adenovirus E4 region, where the protein encoded by the E4 gene is conserved or non-contained. Has conservative amino acid changes and has the same function as wild type (corresponding non-mutated protein), improved function compared to wild type protein, reduced function such as no function compared to wild type protein, or It may be mutated to have a new function compared to the wild type protein or optionally a combination thereof. In one embodiment, the E4 region lacks E4orf4.

  In one embodiment, the E4 region is partially deleted, for example 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45% , 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% are deleted. In one embodiment, the E4 region has a sequence of 32188 bp to 29380 bp of SEQ ID NO: 38.

  In one embodiment, B3 is a bond, ie E4 is absent.

  In one embodiment, B3 has a sequence consisting of 32188 bp to 29380 bp of SEQ ID NO: 38.

  As used herein, a range of numbers includes the endpoint.

  Those skilled in the art will recognize that elements in the formulas herein such as formulas (I), (Ia), (Ib), (Ic), (Id) and (Ie) are adjacent and described herein. It will be understood that the non-coding DNA sequences as well as the gene and coding DNA sequences (structural features) can be embodied. In one or more embodiments, the formulas of this disclosure are intended to describe sequences that occur naturally in the adenovirus genome. In this regard, it will be apparent to those skilled in the art that the formula refers to the major elements that characterize the relevant portion of the genome and is not intended to be an exhaustive description of the genomic stretch of DNA.

  As used herein, E1A, E1B, E3, and E4 are each independently a wild type of each region described herein and its equivalent, a mutant type or a partial deletion type, particularly derived from a known adenovirus Refers to the wild type sequence.

  As used herein, an “insert” refers to a DNA sequence that is incorporated either at the 5 'end, 3' end or within a given DNA sequence reference segment such that it interrupts the reference sequence. The latter is a reference sequence used as a reference point where the insert is located. In the context of this disclosure, the insert is generally present in either SEQ ID NO: 10 or SEQ ID NO: 11. The insert can be either a restriction site insert, a transgene cassette, or both. If the sequence is interrupted, the virus will still contain the original sequence, but generally it will be as two fragments sandwiching the insert.

  In one embodiment, the transgene or transgene cassette does not include an unbiased insertion transposon such as a TN7 transposon or part thereof. A Tn7 transposon as used herein refers to an unbiased insertion transposon as described in WO2008 / 080003.

  In one embodiment, the one or more restriction sites in BX and BY are independently selected from the enzyme specific restriction sites described herein, such as NotI, FseI, AsiSI, SgfI and SbfI, In particular, the inserted restriction sites are different, such as the site specific for NotI located in BX and the site specific for FseI, and SgfI and SbfI located in BY.

  In one embodiment, as described above, regions BX and / or BY do not include a restriction site. Advantageously, the viruses and constructs of the present disclosure can be prepared without restriction sites, for example using synthetic techniques. These techniques allow great flexibility in creating viruses and constructs. Furthermore, the inventors have established that the properties of viruses and constructs are not reduced when they are prepared by synthetic techniques.

Promoter As used herein, a promoter refers to a region of DNA that initiates transcription of a particular gene or genes. Promoters are generally located on the same strand and upstream (ie, 5 ') on DNA, in proximity to the gene they transcribe. Proximal as used in this context means close enough to function as a promoter. In one embodiment, the promoter is within 100 bp of the transcription start site. Thus, an endogenous promoter as used herein refers to a promoter that is naturally present (ie native) in the adenovirus (or construct) into which the transgene has been inserted. In one or more embodiments, the endogenous promoter used is a naturally occurring promoter in the virus that is in its original position in the viral genome, in particular it is used for expression of the transgene or transgene. The primary or only promoter used. In one embodiment, the endogenous promoter used to translate the transgene and optionally promote transcription is one resident, ie, integrated into the adenoviral genome, It was not previously introduced by recombinant technology.

  As used herein, under the control of an endogenous promoter refers to the location where the transgene / transgene cassette is inserted in the proper orientation such that it is under the control of the endogenous promoter. That is, when the promoter is generally on the antisense strand, the cassette is inserted, for example, in the antisense orientation.

  Nevertheless, genes can be expressed in one of two directions. In general, however, for one given (specific) transgene, one direction results in a higher level of expression than the other.

  In one embodiment, the cassette is in the sense direction. That is, the image is transferred in the 5 'to 3' direction. In one embodiment, the cassette is in the antisense orientation. That is, the image is transferred in the 3 'to 5' direction.

  Endogenous promoters in viruses can be utilized, for example, by using genes that encode transgenes and splice acceptor sequences. Thus, in one embodiment, the cassette contains a splice acceptor sequence when under the control of an endogenous promoter. Thus, in one embodiment, the coding sequence, eg, the sequence encoding an antibody or antibody binding fragment, further comprises a splice acceptor sequence.

  In one embodiment, the one or more transgenes or transgene cassettes are under the control of an E4 promoter or major late promoter, such as, for example, a major late promoter (ML promoter).

  As used herein, “under control” means that the transgene is activated, ie transcribed, when directed by a particular promoter.

  As used herein, a major late promoter (ML promoter or MLP) refers to an adenoviral promoter that controls expression of a “late expression” gene, such as the L5 gene. MLP is a “sense strand” promoter. That is, the promoter affects genes that are downstream of the promoter in the 5'-3 'direction. As used herein, the major late promoter refers to the original major late promoter located in the viral genome.

  As used herein, the E4 promoter refers to the adenoviral promoter of the E4 region. Since the E4 region is an antisense region, the promoter is an antisense promoter. That is, the promoter is upstream of the E4 region in the 3'-5 'direction. Thus, any transgene cassette that is under the control of the E4 promoter may need to be properly oriented. In one embodiment, the cassette under the control of the E4 promoter is in the antisense orientation. In one embodiment, the cassette is under the control of the E4 promoter in sense orientation. As used herein, the E4 promoter refers to the original E4 promoter located in the viral genome.

  Thus, in one embodiment, the fiber, hexon and capsid are serotype 11 (such as Ad11p), the viral genome comprises a DNA sequence encoding a therapeutic antibody or antibody binding fragment (such as BiTE), and the transgene is a viral replication The DNA sequence under the control of an adenovirus endogenous promoter selected from the group consisting of E4 and the major late promoter (ie E4 promoter or major late promoter), for example, in particular L5 and E4 Replication-competent oncolytic adenovirus serotype 11 (such as Ad11p) associated with (ie, before or after) the L5 region, such as located after L5 in the viral genome located between Or a viral derivative thereof is provided.

  In one embodiment, the endogenous promoter is introduced at a desired location in the viral genome by recombinant techniques, eg, introduced into a transgene cassette. However, in the context of this specification, this arrangement will generally be referred to as an exogenous promoter.

  In one embodiment, the transgene cassette includes an exogenous promoter. As used herein, an exogenous promoter refers to a promoter that does not naturally occur in the adenovirus into which the transgene is inserted. Typically, the exogenous promoter is from another virus or is a mammalian promoter. As used herein, an exogenous promoter means a DNA element, usually located upstream of a gene of interest, that regulates the transcription of the gene.

  In one embodiment, the regulator of gene expression is an exogenous promoter such as the CMV promoter, such as CMV (cytomegalovirus promoter), CBA (chicken beta actin promoter) or PGK (phosphoglycerate kinase 1 promoter). is there.

  In one embodiment, the CMV exogenous promoter used has the nucleotide sequence of SEQ ID NO: 52. In one embodiment, the PGK exogenous promoter used has the nucleotide sequence of SEQ ID NO: 53. In one embodiment, the CBA exogenous promoter used has the nucleotide sequence of SEQ ID NO: 54.

  In one embodiment, the fiber, hexon and capsid are serotype 11 (such as Ad11p), the viral genome comprises a DNA sequence encoding a therapeutic antibody or antibody binding fragment (such as BiTE according to the present disclosure) and the transgene is a virus. Replication ability, located in a portion of the viral genome that is expressed late in the viral replication cycle so that replication is not interrupted, and the DNA sequence is under the control of an exogenous promoter relative to adenovirus (eg, CMV promoter) Certain oncolytic adenovirus serotypes 11 (such as Ad11p) or viral derivatives thereof are provided. In one embodiment, the DNA sequence encoding the antibody or fragment (such as BiTE according to the present disclosure) is an L5 region, particularly located between the L5 region and the E4 region, as described elsewhere herein. Related to.

  In one embodiment, the exogenous promoter is an antigen presenting cell promoter. As used herein, an antigen presenting cell promoter refers to a promoter of a gene that is selectively expressed by antigen presenting cells such as dendritic cells or macrophages. Such genes include FLT-3, FLT-3 ligand, TLR, CD1a, CD1c, CD11b, CD11c, CD80, CD83, CD86, CD123, CD172a, CD205, CD207, CD209, CD273, CD281, CD283, CD286, CD289, CD287, CXCR4, GITR ligand, IFN-α2, IL-12, IL-23, ILT1, ILT2, ILT3, ILT4, ILT5, ILT7, TSLP receptor, CD141, CD303, CADM1, CLEC9a, XCR1, or CD304; Examples include, but are not limited to, antigen processing and presentation mediators such as CTIIA or GILT. Thus, in one embodiment, an exogenous promoter is suitable for selective expression of a transgene in the antigen presenting cell.

Other regulatory sequences As used herein, a “regulator of gene expression” (or regulator / regulatory element) is a promoter that typically plays a role in gene expression by initiating or enhancing transcription or translation, Refers to genetic features such as enhancer or splice acceptor sequences.

  As used herein, a “splice acceptor sequence”, “splice acceptor” or “splice site” is a regulation that determines when an mRNA molecule is recognized by the micronuclear ribonucleoprotein of the spliceosome complex. Refers to an array. Once assembled, the spliceosome catalyzes splicing between the splice acceptor site and upstream splice donor site of the mRNA molecule and can be translated to produce a single polypeptide or protein. Generate mRNA molecules.

  Different sizes of splice acceptor sequences can be used in the present invention and can be described as short splice acceptor (small), splice acceptor (medium) and branched splice acceptor (large).

  SSA as used herein means a short splice acceptor that typically contains only a splice site, such as just four base pairs. SA as used herein means a splice acceptor, typically comprising 16 base pairs, including a short splice acceptor and a polypyrimidine tract. As used herein, bSA typically refers to a branched splice acceptor comprising a short splice acceptor, a polypyrimidine tract and a branch point, eg, 26 base pairs.

  In one embodiment, the splice acceptor used in the constructs of this disclosure is shown in SEQ ID NOs: 55-57. In one embodiment, the SSA has the nucleotide sequence of SEQ ID NO: 55. In one embodiment, the SA has the nucleotide sequence of SEQ ID NO: 56. In one embodiment, bSA has the nucleotide sequence of SEQ ID NO: 57. In one embodiment, the splice acceptor sequence is independently selected from the group consisting of TGCTAATCTT CCTTTCTCTC TTCAGGG (SEQ ID NO: 57), CCTTTCTCTCTT CAGG (SEQ ID NO: 56), and CAGG (SEQ ID NO: 55).

  In one embodiment, the splice site is immediately advanced (ie, going in the 5 'to 3' direction) by a consensus Scotsack sequence containing CCACC. In one embodiment, the splice site and Kozak sequence are interspersed with up to 100 base pairs. In one embodiment, the Kozak sequence has the nucleotide sequence of SEQ ID NO: 58.

  Typically, when under the control of an endogenous or exogenous promoter (such as an endogenous promoter), there is a Kozak sequence immediately before the coding sequence. The start of the coding region is indicated by the start codon (AUG), for example within the context of the sequence (gcc) gccRccAUGg [SEQ ID NO: 59], and the start of the “start” of the coding sequence is indicated by a bold base. Lower case letters represent bases common to this position (can still vary), upper case letters indicate highly conserved bases, ie the “AUGG” sequence is constant or rarely changes if present . “R” indicates that purine (adenine or guanine) is normally observed at this position, and the sequence in parentheses (gcc) is unclear. Thus, in one embodiment, the start codon AUG is incorporated into the Kozak sequence.

  As used herein, an internal ribosome entry DNA sequence refers to a DNA sequence that encodes an internal ribosome entry sequence (IRES). As used herein, IRES means a nucleotide sequence that allows initiation of translation of an mRNA sequence, including initiation within a messenger RNA (mRNA) sequence. This is particularly useful when the cassette encodes polycistronic mRNA. Using IRES yields polycistronic mRNA that is translated into multiple individual proteins or peptides. In one embodiment, the internal ribosome entry DNA sequence has the nucleotide sequence of SEQ ID NO: 60. In one embodiment, a particular IRES is used only once in the genome. This can be beneficial in terms of genome stability.

  As used herein, “high self-cleaving efficiency 2A peptide” or “2A peptide” refers to a peptide that is efficiently cleaved after translation. Suitable 2A peptides include P2A, F2A, E2A and T2A. The inventors have noted that once a particular DNA sequence encoding a given 2A peptide is used, the same particular DNA sequence may not be used a second time. However, the redundancy of the DNA code can be used to generate a DNA sequence that is translated into the same 2A peptide. The use of 2A peptides is particularly useful when the cassette encodes polycistronic mRNA. With 2A peptides, a single polypeptide chain is translated that is post-translationally modified to produce multiple individual proteins or peptides.

  In one embodiment, the encoded P2A peptide used has the amino acid sequence of SEQ ID NO: 61. In one embodiment, the encoded F2A peptide used has the amino acid sequence of SEQ ID NO: 62. In one embodiment, the encoded E2A peptide used has the amino acid sequence of SEQ ID NO: 63. In one embodiment, the encoded T2A peptide used has the amino acid sequence of SEQ ID NO: 64.

  In one embodiment, the mRNA or each mRNA encoded by the transgene typically includes a polyadenylation signal sequence at the end of the mRNA sequence, eg, as shown in SEQ ID NO: 65. Thus, in one embodiment, the transgene or transgene cassette comprises at least one sequence encoding a polyadenylation signal sequence.

  As used herein, “polyA”, “polyadenylation signal” or “polyadenylation sequence” usually refers to an AATAAA site that can be recognized by a multiprotein complex that, when transcribed, cleaves and polyadenylates a nascent mRNA molecule. Means a DNA sequence comprising

  In one embodiment, the polyadenylation sequence has the nucleotide sequence of SEQ ID NO: 65.

  In one embodiment, the construct does not include a polyadenylation sequence. In one embodiment, the regulator of gene expression is a splice acceptor sequence.

  In one embodiment, the sequence encoding a protein / polypeptide / peptide, such as an antibody or antibody fragment (such as BiTE according to the present disclosure) further comprises a polyadenylation signal.

Molecules encoded by the transgene As described herein, at least one transgene in the virus encodes BiTE, wherein one binding domain is specific for a T cell surface antigen. The second binding domain may target an appropriate antigen, such as a pathogen antigen, cancer antigen, stromal antigen.

Cancer antigens (also referred to as tumor antigens) are one category of particular interest and include, for example, those selected from: CEA, MUC-1, EpCAM, HER receptors HER1, HER2, HER3 , HER4, PEM, A33, G250, carbohydrate antigens Le ^Y , Le ^X , Le ^b , PSMA, TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2, ErbB3, WT1, MUC1, LMP2, Idiotype, HPV E6 and E7, EGFRvIII, HER-2 / neu, MAGE A3, p53 nonmutant, p53 mutant, NY-ESO-1, GD2, PSMA, PCSA, PSA, MelanA / MART1, Ras mutant, Proteinase 3 (PR1), bcr-ab , Tyrosinase, survivin, PSA, hTERT, particularly WT1, MUC1, HER-2 / neu, NY-ESO-1, survivin and hTERT.

  Stromal antigens are described herein such as, for example, FAP, tumor-associated macrophage antigen, and bone marrow-derived suppressor cell antigens such as CD163, CD206, CD68, CD11c, CD11b, CD14, CSF1 receptor, CD15, CD33, and CD66b. Of fibroblast antigens.

  The following target list may be encoded with BiTE according to the present disclosure, if appropriate, or provided as a further therapeutic transgene, or both.

  In one embodiment, the one or more transgenes independently encode proteins such as RNA molecules, peptides, RNA molecules. Advantageously, the transgene can be delivered into the cell and subsequently transcribed and translated if appropriate. Examples of genetic material encoded by the transgene include, for example, antibodies or binding fragments thereof, chemokines, cytokines, immunomodulators, enzymes (eg, capable of converting prodrugs in active agents) and RNAi molecules. .

  As used herein, a peptide refers to an amino acid sequence of 2-50 residues, such as 5-20 residues. As used herein, a polypeptide refers to an amino acid sequence of more than 50 residues that does not include tertiary structure, particularly that does not include secondary and tertiary structure. A protein refers to an amino acid sequence of more than 50 residues having secondary and / or tertiary structure, especially secondary and tertiary structure.

  In one embodiment, the coding sequence encodes a therapeutic RNA, therapeutic peptide, therapeutic polypeptide or therapeutic protein (ie, is a therapeutic gene).

  As used herein, an immunoregulatory gene or transgene refers to a gene that encodes a peptide or protein molecule that can qualitatively or quantitatively modify one or more activities of cells of the immune system. .

  As used herein, a therapeutic gene refers to a gene that encodes an entity that may be useful in the treatment, amelioration, or prevention of a disease, eg, the gene at least slows, stops, or reverses the progression of a disease such as cancer. Expressing a therapeutic protein, polypeptide, peptide or RNA.

  In one embodiment, the entity encoded by the transgene when transcribed or translated in a cell, such as a cancer cell, increases the production of a danger signal by the cell. As used herein, a “danger signal” means, for example, by stimulating cells of the innate immune system and responding directly, but also by acting to enhance cell activation of the adaptive immune system, Refers to various molecules produced by cells undergoing injury, stress or non-apoptotic death that act as warning signals.

  It is known that the tumor microenvironment often changes so that the natural human immune response is down-regulated. Thus, the ability to resume an immune response from within a tumor is potentially very interesting in the treatment of cancer.

  In one embodiment, the encoded therapeutic peptide or protein is designed to be secreted into the extracellular environment. In one embodiment, a functional RNA, peptide, polypeptide or protein such as an antibody is released in the extracellular microenvironment, such as culture supernatant, or in vivo: tissue, stroma, circulation, blood and / or lymphatic system. The

  In one embodiment, the transgene-encoded peptide, polypeptide or protein (including BiTE according to the present disclosure) comprises a signal sequence. A signal peptide as used herein refers to a short 13-36 residue peptide sequence located at the N-terminus of a protein that assists the protein in entry into the secretory pathway for secretion or membrane expression. In one embodiment, the leader sequence (signal peptide) has the amino acid sequence of SEQ ID NO: 66 or 67.

  In another embodiment, the encoded therapeutic peptide or protein, such as an antibody, as a membrane-anchored form on the surface membrane of a cell, eg, by encoding a transmembrane domain at the binding site of the protein or lipid membrane anchor. Designed to express. In general, one or more BiTEs of the present disclosure are not expressed as a cell surface anchor format.

  In one embodiment, a functional RNA, peptide, polypeptide or protein such as an antibody is released from a cell infected with adenovirus, for example by active secretion or as a result of cell lysis. Thus, in one embodiment, adenovirus lyses cells, thereby releasing functional RNA, peptides, polypeptides or proteins such as antibodies.

  In another embodiment, the encoded additional therapeutic peptide or protein, such as an antibody, is designed to be retained in an intact cell.

  Advantageously, a functional RNA, peptide, polypeptide or protein such as an antibody expressed by an adenovirus of the present disclosure can be detected in vivo as both mRNA and antibody protein in tissue. Furthermore, expressed functional RNA, peptides or proteins such as antibodies can bind to their ligands in an ELISA. Still further, functional RNA, peptides, polypeptides or proteins such as antibodies can be detected early (eg, within 3 days of infection) and expression persists over several weeks.

  In one embodiment, an adenovirus of the present disclosure is an antibody such as within about 3 days or more of infection, such as within about 36, 48, 60 or 72 hours, or 2, 3, 4, 5 or 6 days. Expresses any functional RNA, peptide, polypeptide or protein.

  In one embodiment, adenoviruses of the present disclosure express functional RNA, peptides, polypeptides or proteins such as antibodies for several weeks, such as about 1, 2, 3, 4, 5 or 6 weeks. For example, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 days.

  Advantageously, the expression of a functional RNA, peptide or protein such as antibody expression is high enough to be able to detect a functional RNA, peptide, polypeptide or protein such as an antibody in the blood.

  In one embodiment, functional RNA, peptides or proteins, such as antibodies expressed by adenoviruses of this disclosure, enter the bloodstream and / or lymphatic system.

  In one embodiment, the adenovirus of the present disclosure is an oncolytic virus that has an enhanced therapeutic index against cancer cells.

  In one embodiment, the coding sequence further encodes a functional RNA, such as a therapeutic RNA.

  Functional RNA as used herein refers to RNA having a function other than encoding a protein or peptide, for example, RNA suitable for inhibiting or reducing gene activity, including RNAi such as shRNA and miRNA Includes constructs. ShRNA as used herein refers to a short hairpin RNA, which is a sequence of RNA that forms a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). As used herein, miRNA (microRNA) refers to small non-coding RNA molecules (including about 22 nucleotides) that function through base pairing with complementary sequences in mRNA molecules, at the transcription level or transcription. Regulates gene expression at a post-level. The mRNA strands constrained by miRNAs are silenced because they can no longer be translated into proteins by ribosomes, and such complexes are often actively disassembled by cells. Yes.

  In one embodiment, the transgene encodes a protein. Proteins as used herein include protein ligands, protein receptors, or antibody molecules.

  As used herein, protein ligands bind to cellular receptors by, for example, stimulating intracellular signaling and modulating intracellular gene transcription, or otherwise involved to affect cellular function. Refers to the cell surface membrane or secreted protein binding fragment thereof. In one embodiment, the expressed protein is engineered to be expressed on the surface of the cell and / or secreted from the cell.

  In one embodiment, the encoded protein is a bispecific antibody such as BiTE.

  In one embodiment, the transgene further encodes an enzyme, such as an enzyme that aids in the degradation of the tumor's extracellular matrix, such as DNAse, collagenase, matrix metalloproteinase (such as MMP2 or 14), and the like.

  Suitable antibodies and antibody fragments can be agonistic or antagonistic, including those that have anti-cancer activity and those that modify the host cell response to cancer, eg: agonist or antagonist antibodies or antibody fragments Can reduce or normalize tumor angiogenesis. In one embodiment, the agonist antibody or other encoded protein binds to a costimulatory molecule or checkpoint pathway molecule, for example by expressing and attracting and activating antigens, danger signals, cytokines or chemokines, or By enhancing the adaptive immune response, the host cells can be visualized more for the host's innate and adaptive immune response.

  As used herein, a therapeutic antibody or antibody-binding fragment refers to an antibody or antibody-binding fragment that, when inserted into an oncolytic virus, has a beneficial effect on a patient's condition, eg, a cancer being treated.

  A beneficial effect as used herein refers to the desired and / or beneficial effect of an antibody expressed in vivo.

  The classes of therapeutic antibodies and antibody-binding fragments include anti-EGF antibodies, anti-VEGF antibodies, anti-PDGF antibodies, anti-CTLA antibodies, anti-PD1 antibodies, anti-PDL1 antibodies and anti-FGF antibodies.

  Registered therapeutic antibodies suitable for incorporation into the disclosed viruses include abciximab, adalimumab, alemtuzumab (alemtzumab), basiliximab, belimumab, bevacizumab, brentuximab vedotin, canakinumab, cetuximab, certolizumab, certolizumab, certolizumab , Eculzumab, efalizumab, eflizumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, muromonab-CD3, outuzumab, torizumab, torizumab

  In one embodiment, the antibody variable region sequence of the antibody or antibody fragment used has a variable region of bevacizumab (also known as Avastin®) such as 96, 97, 98 or 99% similar or identical to 95- 100% similar or identical.

  Also suitable for incorporation into the viruses of the present disclosure are those antibodies and binding fragments thereof that have been approved for cancer indications such as trastuzumab, tositumumab, rituximab, panitumumab, ofatumumab, ipilimumab, ibritumumab The coding sequence for uxetane, gemtuzumab, denosumab, cetuximab, brentuximab vedotin, avastin and adalimumab.

  In one embodiment, the antibody variable region sequence of the antibody or antibody fragment used is 95-100% similar or identical to the variable region of a known antibody or an antibody disclosed herein.

  As used herein, “antibody molecule” includes antibodies and binding fragments thereof.

  As used herein, an antibody generally refers to a full-length antibody and a bispecific or multispecific format comprising it.

An antibody-binding fragment includes an antibody fragment that can target an antigen with the same, similar, or better specificity as the original “antibody” from which it is derived. Antibody fragments include Fab, modified Fab, Fab ′, modified Fab ′, F (ab ′) ₂ , Fv, single domain antibodies (eg, VH or VL or VHH), scFv, bi-, tri- or tetravalent antibodies, bis -Including scFv, diabody, triabodies, tetrabodies and any epitope-binding fragment of any of the above (eg, Holliger and Hudson, 2005, Nature Biotech. 23 (9): 1126 to 1136; Adair and Lawson, 2005, Drug (See Design Reviews-Online 2 (3), 209-217). Methods for generating and producing these antibody fragments are well known in the art (see, eg, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments for use in the present invention include the Fab and Fab ′ fragments described in International Patent Applications WO2005 / 003169, WO2005 / 003170 and WO2005 / 003171. Multivalent antibodies can include multispecificity, such as bispecificity, or can be monospecific (see, eg, WO92 / 22853, WO05 / 113605, WO2009 / 040562 and WO2010 / 035012). .

  As used herein, specific refers to an antibody or fragment that recognizes only the antigen for which it is specific, or to a specific antigen compared to its binding affinity for a non-specific antigen. Thus, it is intended to refer to an antibody or fragment that has a significantly higher binding affinity, eg, 5, 6, 7, 8, 9, 10 times higher binding affinity.

  Known antibodies or antibody-binding fragments can be used to generate another antibody format with the same CDR or the same variable region, for example, full-length antibodies can be easily converted to Fab, Fab ′ or scFv fragments .

  A wide variety of different forms of antibodies can be used in the constructs of the present disclosure, including antibody molecules derived from non-human animals, human antibody molecules, humanized antibody molecules and chimeric antibody molecules.

  In one embodiment, the antibody or binding fragment is monoclonal. Monoclonal antibodies include hybridoma technology (Kohler & Milstein, 1975, Nature 256: 495-497), trioma technology, human B cell hybridoma technology (Kozbor et al., 1983, Immunology Today, 4:72) and EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapies, pp 77-96, Alan R Liss, Inc., 1985) and the like.

  In one embodiment, the antibody or binding fragment is non-human, ie, completely non-human. This is possible because antibodies and fragments can be delivered into cancer cells by viruses.

  In one embodiment, the antibody is chimeric, eg, has a human constant region and a non-human variable region.

  In one embodiment, the antibody or binding fragment is human, ie, completely derived from humans.

  In one embodiment, the antibody or binding fragment is humanized. Humanized antibodies (including CDR-grafted antibodies) are antibody molecules having one or more complementarity determining regions (CDRs) from non-human species and framework regions from human immunoglobulin molecules (eg, US patents). No. 5,585,089; see WO 91/09967). It will be appreciated that it may only be necessary to transfer the specificity-determining residues of the CDR, but not the entire CDR (see, for example, Kashmiri et al., 2005, Methods, 36, 25-34). A humanized antibody may optionally further comprise one or more framework residues from a non-human species, for example from which the CDRs were derived.

  In one embodiment, the coding sequence encodes an antibody heavy chain, antibody light chain or antibody fragment. As used herein, heavy chain (HC) refers to the large polypeptide subunit of an antibody. Light chain (LC) as used herein refers to the small polypeptide subunit of an antibody. In one embodiment, the antibody light chain comprises either a kappa or lambda CL domain.

  Antibodies for use in the present disclosure can be obtained using any suitable method known in the art. Antigen polypeptides / proteins, including fusion proteins, including cells (recombinant or native) that express a polypeptide (such as activated T cells) are used to produce antibodies that specifically recognize the antigen. be able to. The polypeptide can be a “mature” polypeptide or a biologically active fragment or derivative thereof.

  Antibody screening can be performed using assays for measuring binding to antigen and / or assays for measuring the ability to antagonize receptors. Examples of binding assays are in particular ELISA using a fusion protein (optionally containing a reporter) immobilized on a plate and using a conjugated secondary antibody to detect the anti-antigen antibody bound to the fusion protein. It is.

  When present, the constant region domains of the antibody molecules of the invention can be selected in view of the proposed function of the antibody molecule, particularly the effector functions that may be required. For example, the constant region domain can be a human IgA, IgD, IgE, IgG, or IgM domain. In particular, where the antibody molecule is intended for therapeutic use and antibody effector function is required, human IgG constant region domains, particularly IgG1 and IgG3 isotypes, can be used. Alternatively, IgG2 and IgG4 isotypes may be used where the antibody molecule is intended for therapeutic purposes and antibody effector function is not required, eg, for simple activation or target neutralization. It will be appreciated that sequence variants of these constant region domains may also be used. For example, as described in Angal et al., Molecular Immunology, 1993, 30 (1), 105-108, an IgG4 molecule in which the serine at position 241 is changed to proline can be used.

  For specific antibody functions to deliver activation signals to cells carrying antibody target molecules, such as cells of the immune system, for example, the membrane anchor type of the antibody can be changed so that the antibody is expressed on the surface of the expressing cell. It may be advantageous to use. Such cell surface expressed binding molecules allow efficient multimeric interaction between target signaling molecules on the surface of another cell that enhances delivery of activation signals from the target molecule to the recipient cell. .

  Advantageously, adenoviruses of the present disclosure are capable of expressing full-length antibodies, antibody fragments such as scFv, multispecific antibodies, particularly bispecific antibodies such as BiTE as described herein.

  In one embodiment, the sequence encoding an antibody or antibody fragment (such as BiTE according to the present disclosure) comprises or further comprises an internal ribosome entry sequence. As used herein, an internal ribosome entry sequence (IRES) refers to a nucleotide sequence that allows translation initiation in the middle of a messenger RNA (mRNA) sequence.

  In one embodiment, the encoded therapeutic protein or peptide is a target-specific protein, polypeptide or peptide.

  As used herein, a target-specific protein or peptide is a different protein or peptide that binds directly to the target protein itself, or the level of the target protein or peptide (eg, target-specific) or otherwise modifies. Refers to any of the peptides. The former example is a cytokine, while the latter example is an antibody against that cytokine.

  A target of interest generally relates to a signal transduction pathway associated with a particular cell, cell product, antigen, or disease, particularly cancer. Depending on the context, the target also relates to mRNA transcribed from a gene encoding a protein or polypeptide, or the like, which can be inhibited, for example, by RNAi type technology. Thus, in the context of RNA, such as RNAi technology, the target is the mRNA encoded by the target gene.

  Examples of targets of interest include stimulatory T cell co-receptors and their ligands, checkpoint inhibitory T cell co-receptor molecules and their ligands, receptors and ligands expressed by regulatory T cells, bone marrow-derived suppressor cells and Immunosuppressive immune cells, dendritic cells and antigen presenting cell receptors and their ligands, antigen processing and presentation mediators, cytokines and cytokine receptors, chemokines and chemokine receptors, transcription factors and transcription regulators, intracellular transport molecules and cells Examples include, but are not limited to, functional modulators, tumor cells and tumor microenvironment receptors and products, intracellular tumor cell enzymes such as IDO, and antigens for recognition by immune cells.

  Thus, in one embodiment, a target as used herein refers to a protein or polypeptide that can be, for example, inhibited, neutralized or activated as needed, eg, by an antibody or binding fragment therein. . A target in the context of a cytokine refers to the cytokine itself or an antibody or binding fragment thereof specific for the cytokine. Thus, the virus may encode and express the cytokine itself, as its release can stimulate a “host” immune response. In the context of a ligand, a variant of the ligand can be encoded by a virus that competes with the natural ligand to bind to the receptor. A mutant ligand may have increased binding affinity for the receptor, for example, so that it has a slow dissociation rate, thereby occupying the receptor and increasing or decreasing signaling therefrom. Alternatively, the activity of the mutant ligand can be reduced compared to the wild-type ligand, thereby reducing receptor-mediated binding and overall activity from the natural ligand.

  In one embodiment, a virus or construct according to the present disclosure encodes a prodrug, an immunomodulator and / or an enzyme.

  As used herein, a prodrug refers to a molecule that is administered as an inactive (or not fully active) derivative, which is often converted to an active drug in the body, often through normal metabolic processes. Prodrugs function as a kind of precursor for the intended drug. Prodrug converting enzymes function as enzymes that convert prodrugs to their pharmacologically active forms.

  As used herein, an immunomodulator means a modulator of an immune response. Immunomodulators function in modulating the immune response to a desired level, such as in immune enhancement, immunosuppression, or induction of immune tolerance.

  Two signals are required for T cells to be fully activated. The first signal that is antigen specific is provided via a T cell receptor that interacts with peptide-MHC molecules on the membrane of the antigen presenting cell (APC). The second signal, the costimulatory signal, is non-antigen specific and is provided by the interaction between costimulatory molecules expressed on the APC membrane and T cells. Thus, as used herein, a costimulatory molecule refers to a molecule that provides a signal that is complementary to the antigen-specific signal required by T cells for activation, proliferation and survival. Examples of costimulatory molecules include, but are not limited to, CD28, CD80, CD86, CD83 and 4-1BB.

  As used herein, an enzyme refers to a substance that acts as a catalyst in an organism and regulates the rate at which a chemical reaction proceeds without changing itself in the process.

  The following is a non-exhaustive discussion of exemplary target peptides / polypeptides and proteins.

  In one embodiment, the target is a checkpoint protein, such as an immune checkpoint or cell cycle checkpoint protein. Examples of checkpoint proteins include CTLA-4, PD-1, PD-L1, PD-L2, VISTA, B7-H3, B7-H4, HVEM, ILT-2, ILT-3, ILT-4, TIM- 3, LAG-3, BTLA, LIGHT or CD160, including but not limited to CTLA-4, PD-1, PD-L1 and PD-L2. In one embodiment, an antibody or binding fragment thereof specific for one of them is provided. Thus, in one embodiment, the transgene or transgene cassette encodes an antibody or antibody fragment specific for CTLA-4, PD-1, PD-L1, or PD-L2. In one embodiment, the adenovirus expresses an antibody or antibody fragment specific for CTLA-4, PD-1, PD-L1 or PD-L2.

  In one embodiment, the antibody is a checkpoint inhibitor antibody, such as anti-PD-L1. In one embodiment, the adenovirus expresses a full length anti-human PD-L1 antibody. In one embodiment, expression of the full length anti-human PD-L1 antibody is under the control of an endogenous promoter, such as the major late promoter (MLP), particularly at the BY position. In one embodiment, the adenovirus expresses an scFv form of an anti-human PD-L1 antibody. In one embodiment, the expression of the scFv form of anti-human PD-L1 antibody is under the control of an endogenous promoter, such as the major late promoter, particularly at the BY position.

  In one embodiment, a virus or construct according to the present disclosure is provided that encodes a full-length antibody or an antibody to a scFv specific for CTLA-4 or a binding fragment thereof, as exemplified herein.

  In one embodiment, the target is one or more selected independently from the group consisting of CD16, CD25, CD33, CD332, CD127, CD31, CD43, CD44, CD162, CD301a, CD301b and galectin-3. In one embodiment, an antibody or binding fragment thereof specific to it, such as a full-length antibody or scFv is provided.

  In one embodiment, targets that can be targeted by, for example, an antibody or binding fragment are FLT-3, FLT-3 ligand, TLR, TLR ligand, CCR7, CD1a, CD1c, CD11b, CD11c, CD80, CD83, CD86, CD123, CD172a, CD205, CD207, CD209, CD273, CD281, CD283, CD286, CD289, CD287, CXCR4, GITR ligand, IFN-α2, IL-12, IL-23, ILT1, ILT2, ILT3, ILT4, ILT5, ILT7, TSLP One or more selected independently from the group consisting of receptor, CD141, CD303, CADM1, CLEC9a, XCR1 and CD304.

  In one embodiment, the BiTE target used in the present disclosure is a tumor cell antigen.

In one embodiment, the target is CEA, MUC-1, EpCAM, HER receptor HER 1, HER 2, HER 3, HER 4, PEM, A33, G250, carbohydrate antigens Le ^y , Le ^x , Le ^b , PSMA, One or more independently selected from the group consisting of TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2, and ErbB3.

  In one embodiment, the BiTE target used in the present disclosure is a tumor stromal antigen.

  In one embodiment, BiTE targets used in the present disclosure are FAP, TREM1, IGFBP7, FSP-1, platelet derived growth factor-α receptor (PDGFR-α), platelet derived growth factor β receptor (PDGFR- β) and one or more independently selected from the group consisting of vimentin.

  In one embodiment, the target that can be targeted, for example, by an antibody or binding fragment (such as BiTE) is a cancer target.

  In one embodiment, the target is from the group consisting of OX40, OX40 ligand, CD27, CD28, CD30, CD40, CD40 ligand, TL1A, CD70, CD137, GITR, 4-1BB, ICOS or ICOS ligand, eg CD40 and CD40 ligand. One or more selected independently.

  In one embodiment, the transgene cassette encodes a ligand comprising CD40 or CD40 ligand, or an antibody, antibody fragment or shRNA that targets CD40 or CD40 ligand. In one embodiment, the adenovirus expresses a ligand comprising CD40 or CD40 ligand, or an antibody, antibody fragment or shRNA targeting CD40 or CD40 ligand (specific).

  In one embodiment, the target is IL-1α, IL-1β, IL-6, IL-9, IL-12, IL-13, IL-17, IL-18, IL-22, IL-23, IL- One or more cytokines independently selected from the group consisting of 24, IL-25, IL-26, IL-27, IL-33, IL-35. Interleukin-2 (IL-2), IL-4, IL-5, IL-7, IL-10, IL-15, IL-21, IL-25, IL-1RA, IFNα, IFNβ, IFNγ, TNFα, TGFβ, lymphotoxin α (LTA) and GM-CSF.

  In one embodiment, the transgene cassette is specific for IL-12, IL-18, IL-22, IL-7, IL-15, IL-21, IFNα, IFNγ, TNFα, TGFβ or lymphotoxin α (LTA). An antibody or antibody fragment. In one embodiment, the adenovirus expresses IL-12, IL-18, IL-22, IL-7, IL-15, IL-21, IFNα, IFNγ, TNFα, TGFβ or lymphotoxin α (LTA).

  In one embodiment, the amino acid sequence of IFNγ is SEQ ID NO: 68. In one embodiment, the amino acid sequence of IFNα is SEQ ID NO: 69. In one embodiment, the amino acid sequence of TNFα is SEQ ID NO: 70.

  In one embodiment, the target is a chemokine, for example, IL-8, CCL3, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19, CCL21, CXCR2, CCR2, CCR4, CCR5, CCR6 , CCR7, CCR8, CXCR3, CXCR4, CXCR5 and CRTH2.

  In one embodiment, the transgene cassette encodes an antibody or antibody fragment specific for CCL5, CXCL9, CXCL12, CCL2, CCL19, CCL21, CXCR2, CCR2, CCR4 or CXCR4. In the context of chemokines, targets include locations where the virus encodes and expresses chemokines, eg, to induce or enhance a host immune response against cancer.

  In one embodiment, the adenovirus expresses an antibody or antibody fragment specific for CCL5, CXCL9, CXCL12, CCL2, CCL19, CCL21, CXCR2, CCR2, CCR4 or CXCR4.

  In one embodiment, the target is one or more independently selected from the group consisting of STAT3, STAT1, STAT4, STAT6, CTIIA, MyD88, and NFκB family members, for example, the protein is an inhibitor, such as an antibody or MRNA that is targeted with the binding fragment or transcribed from the related gene is inhibited by mechanisms such as RNAi.

  In one embodiment, the target is a cell survival and death regulator such as HSp70 or survivin, for example the protein is targeted with an inhibitor, such as an antibody or binding fragment thereof, or the mRNA transcribed from the related gene is Inhibited by mechanisms such as RNAi.

  In one embodiment, the target is independent of the group consisting of amphiregulin, BTC, NRG1a, NRG1b, NRG3, TGFα, LRIG1, LRIG3, EGF, EGF-L6, Epigen, HB-EGF, EGFR, Her2, Her3 and Her4. For example, the protein is targeted with an inhibitor, such as an antibody or binding fragment thereof, or the mRNA transcribed from the related gene is inhibited by a mechanism such as RNAi .

  In one embodiment, the target is a ligand or receptor for one or more selected independently from the group consisting of hedgehog, FGF, IGF, Wnt, VEGF, TNF, TGFβ, PDGF and Notch.

  In one embodiment, the adenovirus expresses an antibody or antibody fragment specific for VEGF. In one embodiment, the antibody is an anti-VEGF antibody. For example, an antibody having the amino acid sequence of antibody bevacizumab, or an equivalent thereof. In one embodiment, the adenovirus expresses a full length anti-human VEGF antibody. In one embodiment, expression of the full length anti-human VEGF antibody is under the control of an endogenous promoter, such as the major late promoter (MLP), particularly at the BY position. In one embodiment, the adenovirus expresses an scFv form of an anti-human VEGF antibody. In one embodiment, the expression of the scFv form of anti-human VEGF antibody is under the control of an endogenous promoter, such as a major late promoter, particularly at the BY position.

  In one embodiment, the target is IDO.

  In one embodiment, the target is an antigen for recognition by immune cells (such as a T cell involving BiTE), cytomegalovirus antigen, influenza antigen, hepatitis B virus surface antigen and core antigen, diphtheria toxoid, Crm197. An immunogenic protein from an infectious organism such as tetanus toxoid; a peptide derived from such an antigen that is a known T cell or antibody epitope, or a genetically engineered complex or multimer of such antigen; Tumor-derived protein T cells or peptides derived from such antigens known as epitopes of antibodies; and genetically engineered complexes or such antigens such as WT1, MUC1, LMP2, idiotype, HPV E6 and E7 , EGFRvIII, HER-2 / neu, MA EA3, p53 non-mutated, p53 mutant, NY-ESO-1, GD2, PSMA, PCSA, PSA, gp100, CEA, MelanA / MART1, Ras mutant, proteinase 3 (PR1), bcr-abl, tyrosinase, survivin , PSA, hTERT, in particular WT1, MUC1, HER-2 / neu, NY-ESO-1, survivin or one or more proteins or peptides independently selected from the group consisting of hTERT multimers.

  One skilled in the art will define that there are many possibilities for nucleic acid sequences encoding a given amino acid sequence due to codon redundancy, that tolerant silent nucleobase pair mutations, and any SEQ ID NO. It will be understood that all nucleic acid sequences encoding a given amino acid sequence are contemplated by this disclosure.

  In one embodiment, the peptide, polypeptide or protein encoded by the transgene is a mimotope. A mimotope as used herein is a molecule, often a peptide, that mimics the structure of an epitope. The latter property causes an antibody response similar to that induced by the epitope. An antibody against a given epitope antigen will recognize a mimotope that mimics that epitope. Mimotopes are usually obtained from phage display libraries by biopanning. Vaccines that use mimotopes have been developed. Thus, using antibodies of known specificity to produce libraries (eg, peptide libraries in phage display, such as Ab sequence libraries or non-antibody peptide libraries, particularly peptides having a more stable 3D conformation Can be screened. The production of mimotopes is well described in the art (Tribick G, Rodda S. Combined methods for discovery of peptide ligands witch J to lig. Mashiko T, Ohno Y, Masuko K, Yagi H, Ueima S, Takechi M, Hashimoto Y. Howard therapies to Membranes on Blots. ants expressing target molecules fused to green fluorescent protein.Cancer Sci.2011 102 (1): 25~35 see).

  In one embodiment, a mimotope or other engineered vaccine antigen is encoded by the transgene and expressed to induce an antibody response in the recipient patient, wherein the induced antibody has the desired therapeutic effect. In one embodiment, a GFP-peptide fusion protein having a peptide sequence derived from a desired human ligand is used to induce an anti-self target antibody response. For example, the peptide region of PD-L1, which is known to be important for binding to the target molecule PD-1, includes antibodies whose immune antibody response to the peptide cross-reacts with native PDL1 molecules, and thus PD -L1: can be genetically linked to GFP or other highly immunogenic foreign carrier protein in the same way as encoding an anti-PDL1 antibody directly to help block PD-1 interactions. The concept of a vaccine that induces an anti-self-therapeutic antibody response is well described in the art (Spoh G, Bachmann MF. Therapeutic vaccine to block receptor interactions. Expert Opin Bio 3 Ther. 3: 469-76; Link A, Bachmann MF. Immunodrugs: breaking B-but not T-cell tolerance with therapeutic anti-vaccine A1.Immunotherapies. , Boissier MC.Emerging applications of anticytokine vaccines.Expert Rev Vaccines.2008 7 (10): 1507~17 that of the reference).

  In one or more embodiments, the transgene used encodes the sequence set forth in any one of SEQ ID NOs: 2, 4, 7, 11 or 16.

  In another embodiment, the transgene used encodes a sequence that excludes the C-terminal Deca-His affinity tag, eg, as shown in the virus of any one of SEQ ID NOs: 72-78. To do.

  Advantageously, adenoviruses of the present disclosure express other proteins, such as antibody forms (such as BiTE) and cytokines encoded by transgenes therein, in culture supernatants in vitro or in vivo in tumor tissue Release into the interstitium. The leader sequence can help the encoded protein / polypeptide or peptide exiting the cancer cell. Thus, in one embodiment, the encoded “protein” comprises a leader sequence. As used herein, a leader sequence refers to a polynucleotide sequence located between a promoter sequence and a coding region capable of regulating gene expression at the level of transcription or translation.

  In one embodiment, the coding sequence encodes a peptide. Peptide as used herein refers to an amino acid chain that is not a complete functional protein. Typically, a fragment that is a fragment of the immune system or retains part or all of the function of a protein that can be recognized by the immune system, eg, a peptide of 8 or more amino acids that can be recognized by T cells.

  In one embodiment, the transgene is a reporter gene that encodes an imaging agent including a bioluminescent fluorescent imaging agent (including an activatable fluorescent imaging agent) such as, for example, luciferase, GFP or eGFP or red fluorescent protein.

  As used herein, a reporter gene or reporter sequence refers to a gene or DNA sequence that produces a product that is easily detected in eukaryotic cells and is the other gene to which the DNA is closely linked or bound. It can be used as a marker to determine activity. Reporter genes confer features that are easily identified and measured or selectable markers on the cells or organisms that express them. Reporter genes are often used as an indicator of whether a particular gene has been taken up by or expressed in a cell or biological population. Examples of common reporter genes include LacZ, luciferase, GFP, eGFP, neomycin phosphotransferase, chloramphenicol acetyltransferase, sodium iodide symporter (NIS), nitroreductase (eg NfsA, NfsB) intracellular metallo Examples include, but are not limited to, protein, HSV1-tk or estrogen receptor.

  In one embodiment, the genetic material (especially the transgene) does not encode or express a reporter gene such as a contrast agent, luciferase, GFP or eGFP.

  Viruses according to the present disclosure can be tested for their preference for a particular tumor type by testing their lytic ability in a panel of tumor cells. For example, colon tumor cell lines include HT-29, DLD-1, LS174T, LS1034, SW403, HCT116, SW48, and Colo320DM. Any available colon tumor cell line would be useful for such evaluation as well.

  Prostate cell lines include DU145 and PC-3 cells. Pancreatic cell lines include Panc-1 cells. Breast tumor cell lines include the MDA231 cell line, and ovarian cell lines include the OVCAR-3 cell line. Hematopoietic cell lines include, but are not limited to, Raji and Daudi B lymphocytes, K562 erythroblast-like cells, U937 myeloid cells, and HSB 2 T lymphocytes. Other available tumor cell lines are useful as well.

  The present disclosure also extends to the novel sequences disclosed herein. In one embodiment, the virus has any one of the sequences disclosed herein, eg, any one of SEQ ID NOs: 34-37, or a sequence at least 95% identical thereto, eg, SEQ ID NOs: 79-82. It is shown in the sequence described in any one.

Formulations The present disclosure also relates to pharmaceutical formulations of the virus as described herein.

In one embodiment, a replicable oncolytic liquid parenteral formulation according to the present disclosure is provided, eg, for infusion or injection, wherein the formulation is 1 × 10 ¹⁰ to 1 × 10 ¹⁴ viruses per dose volume. Provide a range of particles.

  A parenteral formulation means a formulation that is designed not to be delivered through the digestive tract. Typical parenteral delivery routes include injection, implantation or infusion. In one embodiment, the formulation is provided in a form for bolus delivery.

  In one embodiment, the parenteral formulation is in the form of an injection. Injection includes intravenous, subcutaneous, intratumoral or intramuscular injection. As used herein, injection means inserting fluid into the body via a syringe. In one embodiment, the disclosed method does not include intratumoral injection.

  In one embodiment, the parenteral formulation is in the form of an infusion.

  As used herein, infusion refers to the administration of fluid at a slower rate by infusion, infusion pump, syringe driver or equivalent device. In one embodiment, the infusion is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35. , 40, 45, 50, 55, 60, 65, 70, 65, 80, 85, 90, 95, 100, 105, 110 or 115 minutes, such as over a period ranging from 1.5 to 120 minutes.

  In one embodiment, one dose of the formulation is less than 100 ml, such as 30 ml, such as when administered by a syringe driver. In one embodiment, one dose of the formulation is less than 10 ml, for example 9, 8, 7, 6, 5, 4, 3, 2 or 1 ml. In one embodiment, one dose of the formulation is less than 1 ml, such as 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1 ml. is there.

  In one embodiment, the injection is administered as a slow injection, eg over 1.5-30 minutes.

  In one embodiment, the formulation is for intravenous (iv) administration. This pathway allows for rapid access to the majority of organs and tissues and is particularly useful for the treatment of established metastases, especially metastases in highly vascularized areas such as the liver and lungs. Therefore, it is particularly effective for delivery of oncolytic viruses.

  Therapeutic formulations will typically be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other parenteral formulation suitable for human administration, formulated as a pre-filled device such as a syringe or vial, particularly as a single dose. Can be

  The formulations generally include a pharmaceutically acceptable diluent or carrier, eg, a non-toxic isotonic carrier that is compatible with the virus and stable for the time required for the virus.

  The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a dispersant or surfactant, such as lecithin, or a nonionic surfactant, such as polysorbate 80 or 40. In the dispersion, maintenance of the required particle size can be aided by the presence of a surfactant. Examples of tonicity agents include sugars in the composition, polyalcohols such as mannitol, sorbitol, or sodium chloride.

  In one embodiment, the parenteral formulation used may include one or more of the following buffers. For example, 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, phosphate buffer and / or Tris buffer, for example sugars such as dextrose, mannose, sucrose, salts such as sodium chloride, magnesium chloride or potassium chloride , Detergents such as nonionic surfactants such as Brigi, PS-80 and PS-40. The formulation may also include preservatives such as EDTA or ethanol or a combination of EDTA and ethanol that are believed to interfere with one or more pathways of possible degradation.

In one embodiment, the formulation is a purified tumor according to the present disclosure, such as 1 × 10 ¹⁰ to 1 × 10 ¹² virus particles per dose, such as 1 × 10 ¹⁰ to 1 × 10 ¹⁴ virus particles per dose. Will contain lytic viruses. In one embodiment, the concentration of virus in the formulation ranges from 2 × 10 ^{8 to} 2 × 10 ¹⁴ vp / mL, such as 2 × 10 ¹² vp / ml.

  In one embodiment, the parenteral formulation comprises glycerol.

  In one embodiment, the formulation comprises an oncolytic adenovirus as described herein, HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), glycerol and a buffer.

  In one embodiment, a parenteral formulation comprises a virus of the present disclosure, eg 5 mM HEPES, eg 5-20% (v / v) glycerol, eg hydrochloric acid to adjust the pH to a range of 7-8, and injection For water.

In one embodiment, 0.7 mL of the disclosed virus at a concentration of 2 × 10 ¹² vp / mL is formulated in 5 mM HEPES, 20% glycerol at a final pH of 7.8.

  A thorough discussion of pharmaceutically acceptable carriers is available at Remington's Pharmaceutical Sciences (Mack Publishing Company, NJ 1991).

  In one embodiment, the formulation is provided as a formulation for topical administration, including inhalation.

  Suitable inhalable formulations include inhalable powders, metered aerosols containing propellant gas, or inhalation solutions without propellant gas. Inhalable powders according to the present disclosure will generally contain the viruses described herein together with physiologically acceptable excipients.

  These inhalable powders include monosaccharides (eg glucose or arabinose), disaccharides (eg lactose, saccharose, maltose), oligosaccharides and polysaccharides (eg dextran), polyalcohols (eg sorbitol, mannitol, xylitol), salts ( For example, sodium chloride, calcium carbonate) or a mixture of each other. The use of monosaccharides or disaccharides, in particular lactose or glucose, is suitably used, but not in the form of their hydrates.

  Particles for deposition in the lung require a particle size of less than 10 microns, for example 1-9 microns, for example 0.1-5 μm, in particular 1-5 μm. The particle size carrying the virus is of utmost importance, so in one embodiment, the virus according to the present disclosure can be adsorbed or absorbed onto particles, such as lactose particles of a given size.

  Propellant gases that can be used to prepare inhalable aerosols are known in the art. Suitable propellant gases are among hydrocarbons such as n-propane, n-butane or isobutane, and halohydrocarbons such as chlorinated and / or fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane. Selected from. The above propellant gases can be used by themselves or in mixtures thereof.

  Particularly suitable propellant gases are halogenated alkane derivatives selected from among TG11, TG12, TG134a and TG227. Of the above halogenated hydrocarbons, TG134a (1,1,1,2-tetrafluoroethane) and TG227 (1,1,1,2,3,3,3-heptafluoropropane) and mixtures thereof are particularly preferred Are suitable.

  The propellant gas-containing inhalation aerosol may also contain other components such as cosolvents, stabilizers, surfactants (surfactants), antioxidants, lubricants and means for adjusting pH. All these ingredients are known in the art.

  The propellant gas-containing inhalation aerosol according to the invention can contain up to 5% by weight of active substance. The aerosol according to the present invention is, for example, 0.002 to 5 wt%, 0.01 to 3 wt%, 0.015 to 2 wt%, 0.1 to 2 wt%, 0.5 to 2 wt%, or 0 0.5 to 1% by weight of active ingredient.

  Alternatively, topical administration to the lung can be achieved, for example, by a nebulizer, such as a nebulizer connected to a compressor (eg, the Pari Master® compressor manufactured by Pari Respiratory Equipment, Inc., Richmond, Va.). LC-Jet Plus® nebulizers) can also be used to administer solution or suspension formulations.

  The viruses of the invention can be delivered dispersed in a solvent, for example in the form of a solution or suspension, for example as already described above for parenteral formulations. It can be suspended in a suitable physiological solution, such as saline or other pharmacologically acceptable solvent or buffer solution. Buffer solutions known in the art provide 0.05 mg to 0.15 mg disodium edetate, 8.0 mg to 9.0 mg per ml water to achieve a pH of about 4.0 to 5.0. It may contain NaCl, 0.15 mg to 0.25 mg polysorbate, 0.25 mg to 0.30 mg anhydrous citric acid, and 0.45 mg to 0.55 mg sodium citrate.

  A therapeutic suspension or solution formulation may also contain one or more excipients. Excipients are well known in the art and include buffers (eg, citrate buffer, phosphate buffer, acetate buffer and bicarbonate buffer), amino acids, urea, alcohol, ascorbic acid, phospholipids, Contains proteins (eg, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensions can be encapsulated in liposomes or biodegradable microspheres. The formulation will generally be provided in a substantially sterile form using aseptic manufacturing methods.

  This involves the manufacture and sterilization of the buffer / solution used in the formulation by filtration, the sterile suspension of the antibody in sterile buffer solution, and the distribution of the formulation into sterile containers by methods well known to those skilled in the art. May be included.

  Nebulizable formulations according to the present disclosure may be provided, for example, as single dose units (eg, sealed plastic containers or vials) packed in a foil envelope. Each vial contains a unit dose, for example in a 2 mL volume of solvent / solution buffer.

In a further aspect of treatment, the present disclosure extends to a virus or formulation thereof as described herein for use in therapy, particularly in the treatment of cancer.

  In one embodiment, the method of treatment is for use in treating a tumor, particularly a solid tumor.

  Tumor, as used herein, is intended to refer to an abnormal mass of tissue that results from excessive cell division that is uncontrolled and progressive, also called a neoplasm. Tumors can be either benign (not cancerous) or malignant. Tumors include all forms of cancer and metastasis.

  In one embodiment, the tumor is a solid tumor. Solid tumors can be localized or metastatic.

  In one embodiment, the tumor is of epithelial origin.

  In one embodiment, the tumor is a malignant tumor such as colorectal cancer, liver cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, thyroid cancer, kidney cancer, bladder cancer, head and neck cancer or lung cancer.

  In one embodiment, the tumor is a colorectal malignancy.

  As used herein, a malignant tumor means a cancerous cell.

  In one embodiment, oncolytic adenovirus is used for the treatment or prevention of metastasis.

  In one embodiment, the methods or formulations herein are used for the treatment of drug resistant cancer.

  In one embodiment, the virus is administered in combination with additional cancer treatment or therapy medications.

  In one embodiment, a virus or formulation according to the present disclosure is provided for use in the manufacture of a medicament for the treatment of cancer, eg, the cancers described above.

  In a further aspect, there is provided a method of treating cancer comprising administering to a patient in need thereof, eg, a human patient, a therapeutically effective amount of a virus or formulation according to the present disclosure.

  In one embodiment, the oncolytic virus or formulation herein is administered in combination with another therapy.

  As used herein, “in combination” is intended to encompass the case where the oncolytic virus is administered before, simultaneously and / or after cancer treatment or therapy.

  Cancer therapy includes surgery, radiation therapy, targeted therapy and / or chemotherapy.

  As used herein, cancer treatment refers to treatment with therapeutic compounds or biological agents, such as antibodies intended to treat cancer and / or maintenance therapy thereof.

  In one embodiment, the cancer treatment is selected from any other anti-cancer therapy including chemotherapeutic agents, targeted anti-cancer agents, radiation therapy, radioisotope therapy, or any combination thereof.

  In one embodiment, a virus of the present disclosure, such as an oncolytic adenovirus, is surgical (neoadjuvant therapy) to shrink tumors, to treat metastases, and / or to prevent metastases or further metastases. It can be used as a pretreatment for such therapy. Oncolytic adenoviruses can be used to treat metastases and / or prevent metastases or further metastases after therapy, such as surgery (adjuvant therapy).

  As used herein, is the administration of additional cancer treatments at the same time or at about the same time as the oncolytic adenovirus formulation. The treatment may be contained within the same formulation or administered as a separate formulation.

  In one embodiment, the virus is administered in combination with a chemotherapeutic agent dose.

  As used herein, chemotherapeutic agents are intended to refer to specific anti-neoplastic chemical agents or drugs that are selectively destructive to malignant cells and tissues. For example, alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. Other examples of chemotherapy include doxorubicin, 5-fluorouracil (5-FU), paclitaxel, capecitabine, irinotecan, and platins such as cisplatin and oxaliplatin. The preferred dose may be selected by the practitioner based on the nature of the cancer being treated.

  In one embodiment, the therapeutic agent is ganciclovir, which can help control the immune response and / or tumor angiogenesis.

  In one embodiment, the one or more therapies used in the methods herein are metronome therapy, i.e., continuous or frequent treatment with low doses of anti-cancer drugs, performed concurrently with other therapies There are many.

  Subgroup B oncolytic adenoviruses, particularly those derived from them such as Ad11 and EnAd, have a mechanism of action almost unrelated to apoptosis and kill cancer cells mainly by the mechanism of epidermal necrosis, Can be particularly synergistic with chemotherapeutic drugs. Moreover, the immunosuppression that occurs during chemotherapy may allow oncolytic viruses to function with higher efficiency.

  The therapeutic dose as used herein is suitable for achieving the intended therapeutic effect when used in an appropriate therapeutic regime, such as an oncolytic adenovirus, which improves the symptoms or condition of the disease, etc. Refers to the amount of virus. A dose can be considered a therapeutic dose in the treatment of cancer or metastasis if the number of virus particles can be sufficient to produce the following results: tumor or metastasis growth slows or stops, or tumor or metastasis is of size It is found to shrink and / or extend the life of the patient. An appropriate therapeutic dose is generally a balance between therapeutic effect and acceptable toxicity, for example where side effects and toxicity are acceptable given the benefits achieved by the treatment.

  In one embodiment, a virus or therapeutic construct (including formulations comprising it) according to the present disclosure is administered weekly, for example, the dose is administered on days 1, 3, 5 for the first week and then weekly thereafter. Be administered.

  In one embodiment, a virus or therapeutic construct (including formulations comprising it) according to the present disclosure is administered twice or three times a week, for example, once every week on days 1, 3 and 5 The second or third week is also administered on the first, third and fifth days. This dosage regimen can be repeated as many times as necessary.

  In one embodiment, a virus or therapeutic construct (including formulations comprising it) according to the present disclosure is administered monthly.

  In one embodiment, the viruses and constructs of the present disclosure are prepared by recombinant techniques. One skilled in the art will appreciate that the armed adenovirus genome can be produced by other technical means including fully synthesizing a plasmid containing the genome or a portion of the entire genome. One skilled in the art will understand that when synthesizing a genome, the insertion region may not contain restriction site nucleotides since the latter is an artifact after insertion of the gene using cloning methods.

  In one embodiment, the armed adenovirus genome is produced completely synthetically, eg, according to SEQ ID NOs: 34-37.

  The disclosure herein further extends to an adenovirus of formula (I) or a sub-formula thereof obtained or obtainable from inserting a transgene or transgene cassette.

  As used herein "is (Is)" means including.

  In the context of this specification, “comprising” should be interpreted as “including”.

  Embodiments of the invention that include specific features / elements are intended to cover alternative embodiments that “consist” or “consist essentially of” the relevant elements / features.

  Embodiments of the present invention can be combined where technically appropriate.

  Technical references such as patents and applications are hereby incorporated by reference.

  Any embodiment specifically and explicitly recited herein may form the basis for a disclaimer, alone or in combination with one or more further embodiments.

  This application claims priority from GB1614607.8, GB1700663.6, GB17062119.1 and GB1713765.4, which is incorporated herein by reference. These documents can be used to correct errors in the specification, in particular errors in the sequence listing.

  The invention will be further described, by way of example only, in the following examples with reference to the accompanying drawings.

(A) Schematic of a BiTE antibody of the present disclosure comprising or lacking any decahistidine affinity tag. Ig SP: signal peptide. 10His: Decahistidine affinity tag. L: GS linker. V _L : Variable optical domain. _VH variable heavy chain domain. (B) Plasmid map of pSF-CMV-EpCAMBiTE. (C) Plasmid map of pSF-CMV-FAPBiTE. (D) Plasmid map of pSF-CMV-control BiTE. Same as above. Same as above. Same as above. (A) Dot blot showing quantification of recombinant BiTE. (B) is a graph showing ELISA results for FAP. (C) Graph showing ELISA results for EpCAM. Same as above. Graph showing CD69 (A) and CD25 (B) expression levels for T cells co-cultured with NHDF cells alone or in the presence of FAP BiTE and control BiTE measured using flow cytometry . Same as above. (A) Graph showing the expression level of IFNγ for T cells, co-cultured with NHDF cells, alone or in the presence of FAP BiTE and control BiTE, as measured by intracellular cytokine staining. (B) and (C) Graph showing CD69 and CD25 expression levels for T cells co-cultured with DLD cells alone or in the presence of EpCAM BiTE and control BiTE, measured using flow cytometry. Same as above. (A) Graph showing the expression level of IFNγ for T cells co-cultured with DLD cells in the presence of EpCAM BiTE and control BiTE as measured by intracellular cytokine staining. (B) and (C) Graph showing CD69 and CD25 levels for PBMC co-cultured with DLD cells in the presence of EpCAM BiTE and control BiTE as measured by flow cytometry. Same as above. (A) Graph showing the results of LDH assay showing cytotoxicity of NHDF cells co-cultured with T cells and FAP BiTE or control BiTE. (B) Graph showing the results of LDH assay showing cytotoxicity of BTC100 cells co-cultured with T cells and FAP BiTE or control BiTE. (C) Images of NHDF cells after co-culture of T cells and FAP BiTE versus control BiTE. (A) Scatter plot showing FAP expression in multiple patient-derived cells. (B) Graph showing% of cells expressing EpCAM and FAP across multiple cells and cell lines. Same as above. Same as above. (A) Graph showing NHDF dose response for increasing BiTE concentrations of FAP BiTE. (B) and (C) Graph showing the results of LDH assay showing cytotoxicity of TLD cells and DLD cells co-cultured with EpCAM BiTE or control BiTE. Same as above. (A) Graph showing the results of LDH assay showing cytotoxicity of SKOV cells co-cultured with T cells and EpCAM BiTE or control BiTE. (B) Graph showing the results of LDH assay showing cytotoxicity of MCF7 cells co-cultured with T cells and EpCAM BiTE or control BiTE. Same as above. Graph showing NHDF dose response for increasing BiTE concentrations of EpCAM BiTE. (A) Graph showing FAP expression in CHO cells determined by FAP or isotope control antibody and analyzed by flow cytometry. (B) A graph showing the results of LDH assay showing cytotoxicity of CHO or CHO-FAP cells co-cultured with T cells and FAP BiTE or control BiTE. Graph showing T cell activation (based on CD69 and CD25 expression levels) by CHO versus CHO-FAP cells analyzed using flow cytometry. (A) Graph showing EpCAM expression of the parental cell line versus the stable transfected mutant as determined by staining with EpCAM or an isotopic control antibody and analyzing using flow cytometry. (B) Graph showing the results of LDH assay showing cytotoxicity of CHO or CHO-EpCAM cells co-cultured with T cells and EpCAM BiTE or control BiTE. Same as above. Graph showing T cell activation (based on CD69 and CD25 expression levels) by CHO vs. CHO-EpCAM cells analyzed using flow cytometry. (A) Graph showing the ability of FAP BiTE to activate CD4 + or CD8 + T cells (based on CD69 and CD25 expression levels) analyzed using flow cytometry. (B) Graph showing the results of LDH assay showing cytotoxicity of NHDF cells co-cultured with CD4 + or CD8 + T cells and FAP BiTE or control BiTE. (A) Graph showing CD4 + and CD8 + T cell activation (based on CD69 and CD25 expression levels) by DLD cells in the presence of EpCAM or control BiTE, analyzed using flow cytometry. (B) Graph showing the results of LDH assay showing cytotoxicity of CD4 + or CD8 + T cells and DLD cells co-cultured with EpCAM BiTE or control BiTE. (A) Graph showing the number of ascites-derived CD3 + T cells cultured in control or FAP BiTE. (B) A graph showing the CD25 expression level of ascites-derived T cells cultured in control or FAP BiTE. (C) A graph showing the number of FAP + cells derived from ascites cultured in control or FAP BiTE. (A) Schematic of the adenovirus genome of the present disclosure. (B) Graph comparing the kinetics of CMV versus SA promoter driven expression. (A) Graph showing the quantification of the number of detected viral genomes per cell for NG-601, NG-602, NG-603, NG-604, NG-605, NG-606 and EnAd. (B) A graph showing the oncolytic activity of NG-601, NG-602, NG-603, NG-604, NG-605, NG-606 or EnAd evaluated by infection of A549 cells. (A) T cells by NG-601, NG-602, NG-605 and NG-606 (based on expression levels of CD69 and CD25) when co-cultured with CHO-FAP analyzed using flow cytometry The graph which shows activation. (B) T cell activity by NG-601, NG-602, NG-605 and NG-606 (based on CD69 and CD25 expression levels) when co-cultured with CHO-EpCAM, analyzed using flow cytometry A graph showing crystallization. Same as above. The graph which shows the result of the experiment for determining the quantity of FAP BiTE produced from NG-605 and NG-606. The graph which shows the result of the experiment for determining the quantity of EpCAMBiTE produced from NG-601 and NG-602. Microscopic images of Ad293 cells infected with NG-607, NG-608, NG-609 and NG-610. (A) Graph showing cytotoxicity of DLD cells infected with EnAd, analyzed using XCELLligence. (B) Graph showing the cytotoxicity of SKOV cells infected with EnAd, analyzed using XCELLligence. (C) Graph showing the cytotoxicity of NHDF cells infected with EnAd, analyzed using XCELLligence. (A) Graph showing the ability of NG-603, NG-604, NG-605, NG-606 and EnAd to kill NHDF cells, analyzed using XCELLligence. (B) Graph showing the ability of NG-603, NG-604, NG-605, NG-606 and EnAd to kill NHDF cells analyzed using the LDH assay. T cell activation (based on CD69 and CD25 expression levels) by NG-603, NG-604, NG-605, NG-606 co-cultured with NHDF cells, SKOV and T cells analyzed using flow cytometry Graph showing. (A) by NG-603, NG-604, NG-605, NG-606 co-cultured with NHDF and SKOV cells versus SKOV alone (based on expression levels of CD69 and CD25) analyzed using flow cytometry Graph showing T cell activation. (B) Graph showing the cytotoxicity of NHDF cells infected with NG-605 and NG-606 analyzed using LDH assay. Still frame from time lapse video of lysis of NHDF cells by recombinant FAP BiTE, EnAd, NG-603 or NG-605. Still frame from time-lapse video of lysis of NHDF cells by NG-607, NG-608, NG-609 or NG-610. Shows the cytotoxicity of DLD cells infected with EnAd, NG-601, NG-602, NG-603 and NG-604 in the presence or absence of T cells, as analyzed using XCELL Ligence Graph. Cytotoxicity of DLD cells infected with EnAd, NG-601, NG-602, NG-603 and NG-604 in the presence or absence of T cells, analyzed using the LDH assay Graph showing. Graph showing T cell activation (based on CD69 and CD25 expression levels) by EnAd, NG-601, NG-602, NG-603 and NG-604 analyzed by flow cytometry. To determine the ability of NG-601 to kill DLD tumor cells by varying the multiplicity of infection (MOI) in the presence or absence of CD3 ⁺ T cells, as assessed using xCELLligence results of the experiment. Same as above. Same as above. Graph showing the ability of EnAd and NG-601, NG-602, NG-603, and NG-604 to kill SKOV tumor cells in the presence or absence of CD3 ⁺ T cells, evaluated using xCELLligence. Graph showing the ability of EnAd and NG-601, NG-602, NG-603, and NG-604 to kill SKOV tumor cells in the presence or absence of CD3 ⁺ T cells, as assessed using the LDH assay. T cell activation (based on expression levels of CD69 and CD25) by EnAd, NG-601, NG-602, NG-603 and NG-604 co-cultured with SKOV tumor cells, analyzed using flow cytometry Graph showing. Shows T cell activation (based on expression levels of CD69 and CD25) by EnAd, NG-601, NG-602, NG-603 and NG-604 co-cultured with ascites cells analyzed using flow cytometry Graph. Still frame from time-lapse video of lysis of NHDF cells by EpCAM BiTE, EnAd, NG-601 or NG-603. Microscopic image of ascites cells obtained from a patient infected with the virus of the present disclosure and stained with EnAd-CMV-GFP and EnAd-SA-GFP as a reporter for determining infection and late viral gene expression. (A) Graph showing the expression level of CD25 on CD3 + T cells in ascites samples infected with the virus of the present disclosure. (B) Graph showing the number of FAP + cells in ascites samples infected with the virus of the present disclosure. Microscopic images of ascites cells obtained from cancer patients infected with the virus of the present disclosure and stained with EnAd-CMV-GFP and EnAd-SA-GFP as reporters to determine infection and late viral gene expression. 3 is a graph showing the number of CD3 + T cells in ascites samples obtained from cancer patients infected with the virus of the present disclosure. FIG. 3 is a graph showing the expression level of CD25 on CD3 + T cells in ascites samples obtained from cancer patients infected with the virus of the present disclosure. FIG. 3 is a graph showing the number of FAP + cells in ascites samples obtained from cancer patients infected with the virus of the present disclosure. Characterization of EpCAM BiTE and its effect on PBMC-derived T cells (A) Schematic structure of BiTE targeting EpCAM and non-specific control BiTE. The VL and VH domains are linked to a flexible peptide linker (L) rich in serine and glycine for flexibility and solubility. Ig SP, light chain immunoglobulin signal peptide. 10His, decahistidine affinity tag. (B) Induction of activation markers CD69 and (C) CD25 on CD3 purified PBMC cultured alone or with DLD cells (5: 1) in the presence of BiTE-containing supernatant. CD69 and CD25 were measured by flow cytometry after 24 hours of co-culture. Significance was assessed compared to IgG isotype. (D) Percentage of IFNγ positive T cells after 6 hours in co-culture with DLD cells (5: 1) and BiTE-containing supernatant. (E) Mitosis index of CFSE-stained T cells co-cultured with DLD cells (5: 1) and BiTE-containing supernatant and proliferation expressed as a percentage of the parent T cell population entering proliferation. Fluorescence was measured by flow cytometry after 5 days of co-culture. The mitotic index was modeled using the FlowJo proliferation tool. (F) T-cell degranulation measured by expression of CD107a co-cultured with DLD cells (5: 1) and BiTE-containing supernatant. Expression was assessed by 6 hour co-culture with CD107a specific antibody followed by flow cytometric analysis. (G) Supernatants from co-cultures of T cells and DLD cells (5: 1) were used for 48 hours in the presence of BiTE-containing supernatants to measure cytokine levels with the LEGENDplex human Th cytokine panel. Each condition was measured in a biological triplicate and data were expressed as mean ± SD. Significance was assessed compared to untreated using a one-way analysis of variance test with Tukey's Post Hoc analysis unless otherwise stated. * P <0.05, ** p <0.01, *** p <0.001 Same as above. Same as above. Same as above. Same as above. Characterization of recombinant EpCAM BiTE (A) Dot blot to estimate the amount of EpCAM BiTE produced by transfected HEK293A cells. (B) ELISA measuring the level of EpCAM binding by control or recombinant EpCAM or non-specific BiTE. Significance was assessed by comparing to an empty vector control sample using a one-way analysis of variance test using Tukey's Post Hoc analysis. *** p <0.001 Evaluation of antigen specificity of T cell cytotoxicity via EpCAM BiTE (A) Co-cultured with CHO or CHO-EpCAM cells (5: 1) and BiTE-containing supernatant measured by FACS analysis after 24 hours of co-culture. Induction of activation marker CD25 on cultured CD3 + T cells. (B) Cytotoxicity of CHO or CHO-EpCAM cells cultured only in BiTE-containing supernatant or in co-culture with T cells. Cytotoxicity was assessed by the release of LDH into the culture supernatant after 24 hours of incubation. (C) Cytotoxicity of multiple EpCAM positive cancer cells 24 hours after co-culture with T cells (1: 5) and BiTE-containing supernatant. Viability was measured by MTS assay 24 hours after co-culture. (D) EpCAM expression level assessed by FACS analysis of EpCAM positive cell line of (C) compared to background fluorescence measured using isotype control antibody (N = 1). (AC) Each condition was measured by biological triple measurement and expressed as mean ± SD. Significance was assessed using a one-way analysis of variance test using Tukey's Post Hoc analysis compared to untreated or T cell only controls. * P <0.05, ** p <0.01, *** p <0.001 Same as above. EnAd Expression in SKOV3 Cells EpCAM BiTE Cytotoxicity SKOV3 cells are incubated with EnAd or recombinant virus in the absence (A) or presence (B) of T cells to determine cytotoxicity at a specific time point in LDH Measured by release. Significance was assessed by comparing to uninfected control wells using a one-way analysis of variance test using Tukey's Post Hoc analysis. *** p <0.001 Identification of which T cells are involved in BiTE-mediated cytotoxicity (A) CD4 and CD8 cells after 24 hours co-culture of CD3 T cells with DLD cells (5: 1) and BiTE-containing supernatants Activation of T cells via BiTE. Activation was assessed by surface expression of CD69 and CD25 and measured by flow cytometry. (B) Proliferative response in co-culture of CFSE stained CD4 and CD8 T cells with DLD cells and incubation with BiTE containing supernatant. After 5 days of incubation, fluorescence was measured by FACS analysis. (C) Degranulation of CD4 and CD8 cells after co-culture with DLD cells and BiTE-containing supernatant for 6 hours. During co-culture, CD107a specific antibody is added to the medium and degranulation is assessed by flow cytometry. (D) Cytotoxicity by CD4 or CD8 T cell subsets is assessed by LDH release into the supernatant after incubation of DLD cells with CD4 or CD8 purified T cells (1: 5) and BiTE containing supernatant for 24 hours. Is done. Each condition was measured in a biological triplicate and expressed as mean ± SD. Significance was assessed using a one-way analysis of variance test with Tukey's Post Hoc analysis, comparing EpCAM BiTE treatment to control BiTE unless otherwise stated. * P <0.05, ** p <0.01, *** p <0.001 Same as above. Same as above. Cytotoxicity and T cell activation by EnAd-expressing EpCAM BiTE in DLD cells Infecting DLD cells in the absence (A) or presence (B) of infected DLD cells and infecting with T cells Cultured and cytotoxicity was measured by LDH release at specific time points. (C to D) T cells were harvested from (B), stained with activation markers CD69 (C) or CD25 (D) and analyzed by flow cytometry. (EF) Quantification of EpCAM BiTE produced from DLD cells infected with recombinant virus. Standard curve (E) of LDH release (Ab) of DLD in co-culture with CD3 + cells and various known amounts of recombinant EpCAM BiTE. In parallel, the co-cultures were incubated with diluted supernatants (10,000 times) from DLD cells infected for 3 days (F). The standard curve allowed an approximate measurement of EpCAM BiTE produced in 165 μg and 50 μg of DLD cells per million for EnAd-CMV-EpCAMBiTE and EnAd-SA-EpCAMBiTE, respectively. Significance was assessed by comparing to uninfected control wells using a one-way analysis of variance test using Tukey's Post Hoc analysis. *** p <0.001 Same as above. Same as above. Same as above. Characterization of the oncolytic virus EnAd expressing EpCAM BiTE using cell lines and PBMC-derived T cells (A) DLD cells infected with parental EnAd or recombinant virus (100 vp / cell) and wells at 24 or 72 hours Was recovered. Replication was assessed by measuring the genome using qPCR against viral hexons. (B) Cytotoxicity of DLD cells infected with EnAd or recombinant virus with increasing concentrations of virus. Cytotoxicity was measured by MTS assay 5 days after infection. (C) Supernatants from uninfected or virus infected HEK293A cells on day 3 were evaluated for transgene expression by immunoblot analysis and probed with anti-His antibody. (D) Induction of CD25 positive T cell activation marker CD25 cultured with CHO or CHO-EpCAM (E: T 5: 1) and diluted HEK293A supernatant of (D). Activation was measured by surface expression of CD25 by flow cytometry. (E) Cytotoxicity of CHO or CHO-EpCAM cells incubated with HEK293A supernatant of (D) alone or by co-culture with CD3 purified PBMC (E: T 5: 1). HEK293A supernatant was diluted 300-fold. Cytotoxicity was assessed by LDH released into the supernatant after 24 hours incubation. Each condition was measured in a biological triplicate and expressed as mean ± SD. Significance was assessed using a one-way analysis of variance test using Tukey's Post Hoc analysis at each condition compared to untreated. * P <0.05, ** p <0.01, *** p <0.001 Same as above. Same as above. Same as above. Intracellular composition of malignant exudates (A) Representative images showing ascites and exudate screening for their cellular composition as assessed by flow cytometry (pleural fluid sample, patient 3 in FIG. 57). (B) The absolute number of each cell type (in a 10,000 cell sample size) is listed in the table. Same as above. The superior potency of EnAd expressing EpCAM BiTE (AB) SKOV3 cells in a partially EnAd resistant cancer cell line was monitored in real time over 160 hours by an xCELLligence based cytotoxicity assay. SKOV3 cells were infected by inoculating with EnAd virus armed with EnAd or BiTE at 0 hours, and uninfected cells were used as negative controls. (B) CD2 purified PBMC (5: 1) was added 2 hours after infection and impedance was measured at 15 minute intervals. (CD) CD3-purified PBMC were cultured with SKOV3 cells (5: 1) infected with parental EnAd or recombinant armed virus. At each time point, T cells were harvested and analyzed for surface expression of CD69 (C) or CD25 (D) by flow cytometry. (E) Co-culture of EnAd, EnAd-CMVEpCAMBiTE infected or uninfected, SKOV3 cancer cells (unstained), NHDF fibroblasts (red), and CD3 purified PBMC (blue). The time lapse sequence shown. Apoptosis was visualized using CellEvent Caspase 3/7 detection reagent (green). Images were taken for 96 hours at 15 minute intervals with a Nikon TE 2000-E Eclipse inverted microscope. A representative image was recorded at the time displayed. Original magnification x10. Scale bar 100 μm. (AD) Each condition was measured in biological triplicate and expressed as mean ± SD. Significance was assessed by comparison with uninfected controls using a one-way analysis of variance test using Tukey's Post Hoc analysis. * P <0.05, ** p <0.01, *** p <0.001 Same as above. Same as above. Effect of PD1 antibody on PD1 expression and BiTE-mediated T cell activation (A) PD1 expression by endogenous T cells after initial isolation from pleural effusion was assessed by flow cytometry. (BD) Unpurified whole cells from pleural effusion (from 3 different patients) were incubated in 100% liquid from the same pleural effusion in the presence of free BiTE, EnAd, or recombinant virus. After 5 days, the total cell population was collected and the number of (B) CD3 + T cells and (C) CD25 + was quantified. (D) The number of EpCAM + cells was measured using flow cytometry. Significance was assessed by comparing to untreated control wells using a one-way analysis of variance test using Tukey's Post Hoc analysis. *** p <0.001 Same as above. EnAd expressing EpCAM BiTE can first be isolated from the ascites of three patients that can selectively kill primary human tumor cells of patients who have received pre-chemotherapy, grown ex vivo, and then combined (A) Cytotoxicity of EpCAM + cells, or (B) FAP + fibroblasts, incubated with replacement BiTE or infected with EnAd or recombinant virus. Cytotoxicity was measured by flow cytometry after 5 days. (C) Induction of activation marker CD25 on CD3-positive T cells cultured with EpCAM + and FAP + cells from (A + B) ascites. Each condition was measured in a biological triplicate and expressed as mean ± SD. Significance was assessed by comparison to untreated using a one-way analysis of variance test with Tukey's Post Hoc analysis. * P <0.05, ** p <0.01, *** p <0.001 Same as above. EpCAM BiTE overcomes the immunosuppressive effect of ascites and activates endogenous T cells (AB) PBMC-derived T cells in RPMI medium or in the presence of 100% peritoneal ascites from five ovarian cancer patients Incubated with anti-CD3 antibody. (A) T cell activation marker induction CD69 and CD25 was analyzed at 24 hours, and (B) T cell degranulation was measured by CD107a expression using flow cytometry. (C) The viability of MCF7 cells was monitored in real time over 60 hours by an xCELLligence based cytotoxicity assay. MCF7 cells were seeded and incubated with control or EpCAM BiTE for 25 hours in the presence of RPMI medium or 100% ascites # 1 or # 2. Untreated cells were used as a negative control. CD3 purified PBMC (5: 1) was added simultaneously and impedance was measured at 15 minute intervals. (D) Endogenous unpurified total cells from peritoneal ascites were incubated in 100% ascites in the presence of free EpCAM or control BiTE. After 24 hours, the total cell population was collected and the number of CD3 + / CD69 + and CD3 + / CD25 + cells was determined by flow cytometry. Each condition was measured in a biological triplicate and expressed as mean ± SD. Significance was assessed using a one-way analysis of variance test using Tukey's Post Hoc analysis by comparison with RPMI (A + B), untreated (D) or control BiTE (E). * P <0.05, ** p <0.01, *** p <0.001 Same as above. Same as above. Same as above. EnAd expressing EpCAM BiTE can activate endogenous T cells, killing endogenous tumor cells in malignant pleural effusions, and unpurified whole cells from pleural effusions (from 4 different patients) Incubated in 100% fluid from the same pleural effusion in the presence of free BiTE, EnAd or recombinant virus. After 5 days, the total cell population was collected and the number of (A) CD3 + T cells and (B) CD25 + was quantified. (C) The number of EpCAM + cells was measured using flow cytometry. (D) Representative image (magnification × 10, scale bar 100 μm) and flow cytometric analysis of patient 3 pleural effusion cells (cancer cells and lymphocytes) and flow cytometry analysis (E) day 5 after treatment with EnAd or EnAd-CMVEpCAM BiTE In addition, cytokine levels were measured by the LEGENDplex human Th cytokine panel using pleural effusion cultures after incubation with free recombinant BiTE or infection with EnAd or recombinant virus. Each condition was measured in a biological triplicate and expressed as mean ± SD. Significance was assessed by comparing to untreated control samples using a one-way analysis of variance test using Tukey's Post Hoc analysis. * P <0.05, ** p <0.01, *** p <0.001 Same as above. Same as above. Same as above. The amount of IL-10 measured in normal serum (NS) or patient's malignant exudate (A: peritoneal ascites, P: pleural effusion) using a human IL-10 ELISA MAX kit (Biolegend, 430604). CD3 / 28 bead-mediated PBMC T cell activation versus normal serum (based on the level of CD69 / CD25) in patient fluid as measured by flow cytometry. A: Patient exudate, P: pleural effusion. CD3 / 28 bead-mediated PBMC T cell degranulation (based on expression of CD107a) in patient body fluids. A: Ascites, P: Pleural effusion. Correlation between IL-10 levels in patient body fluids and during CD3 / CD28 bead-mediated T cell degranulation. EpCAM BiTE bead-mediated PBMC T cell activation (based on expression of CD69 / CD25) in patient bodily fluids. A: Ascites, P: Pleural effusion. EpCAM BiTE bead-mediated PBMC T cell degranulation (based on the expression of CD107a) in patient bodily fluids. A: Ascites, P: Pleural effusion. EpCAM BiTE bead-mediated cytotoxicity of SKOV3 in patient bodily fluids. A: Ascites, P: Pleural effusion. EpCAM BiTE-mediated T cell activation (based on expression of CD25 / CD69) in RPMI medium versus ascites. The ability of EnAd-SA-EpCAMBiTE and EnAd-SA-ControlBiTE to induce T cell-mediated lysis of target cells in RPMI medium versus ascites. ((A) Number of CD3 +. (B) CD25 expression of T cells. (C) Number of EpCAM + cells determined by flow cytometry. Same as above. Same as above. The ability of EnAd-SA-EpCAMBiTE and EnAd-SA-ControlBiTE to induce T cell-mediated target cell lysis in ascites (7 patient samples). (A) Number of CD3 +. (B) CD25 expression of T cells. (C) Number of EpCAM + cells determined by flow cytometry. See FIG. 67A for a description. Same as above. Same as above. Comparison of activation of T cell cytokine production by recombinant FAP BiTE protein in the presence of human fibroblasts and by polyclonal activation with anti-CD3 / CD28 beads. (A) IFNγ levels measured by ELISA. (B) Cytokine levels measured by cytokine bead array. BiTE targeting FAP induces degranulation of T cells and specific cytotoxicity of FAP + cells (A) NHDF cells (5: 1) and degranulation of T cells in culture, and (B) BiTE Containing supernatant. Degranulation was assessed by expression of CD107a after 6 hours of incubation with CD107a specific antibodies and measured by flow cytometry. CD3 / CD28 Dynabeads were used as a positive control. (C) NHDF cell cytotoxicity after 24 hours in co-culture with T cells (1: 5) and 10-fold serial dilutions of BiTE-containing supernatant. Cytotoxicity was assessed by the release of LDH into the culture supernatant. (D) Lysis of NHDF by LDH release (left) and CD25 induction on T cells (right) with 24 healthy donors and PBMC-derived T cells from BiTE-containing supernatants (1: 5) Evaluation was performed after time co-culture. Same as above. EnAd expressing FAP BiTE selectively kills FAP ⁺ fibroblasts and reduces TGFb in peritoneal ascites samples (A, B) 72 hours of PBMC-derived T cells and EnAd or recombinant virus Number of FAP ⁺ fibroblasts (A) and EpCAM ⁺ tumor cells (B) after culture. Ascites cells were first isolated from the ascites of three patients and expanded ex vivo. Cell number was determined by flow cytometry 72 hours after infection. (C) Induction of activation marker CD25 on PBMC-derived CD3 cells from (A) was measured 72 hours after infection. (D) The level of TGFb was measured by ELISA using the supernatant recovered from (A). Same as above. Endogenous tumor-associated T cell activation and associated FAP + cell death in malignant ascites biopsy samples of patients with FAP BiTE protein and EnAd-FAPBiTE virus. (A) T cell activation measured by CD25 expression. (B) Remaining number of FAP + cells measured by flow cytometry. Effect of PD-L1 to block antibodies against BiTE-mediated T cell activation in patient samples (A) PD1 expression by PD-L1 on endogenous T cells and FAP + cells after their initial isolation from peritoneal ascites Were evaluated by flow cytometry. (B) Unpurified whole cells from peritoneal ascites in 50% fluid from the same exudate in the presence of free BiTE, EnAd or recombinant virus, with or without anti-PD-L1 blocking antibody Incubated with. Two days later, the total cell population was collected and the number of CD25 + T cells was quantified by flow cytometry. (C) The amount of interferon gamma in the culture supernatant from (B, D) was measured by ELISA. (D) The number of remaining FAP + cells in (B) was measured using flow cytometry. Same as above. EnTe expressing BiTE activates T cells from patient biopsy samples and redirects to lyse NHDF fibroblasts (A) after isolation from healthy donor or malignant exudate cancer biopsy samples PD-1 expression by endogenous T cells. PD-1 expression was measured by flow cytometry. (B) Proportion of CD3 ⁺ cells in unpurified PBMC cell population and cancer biopsy samples measured by flow cytometry. (C) Interferon gamma level measured by ELISA in the culture supernatant collected from (B) 120 hours after treatment. (D) Survival of NHDF fibroblasts was monitored in real time over 130 hours by xCELLiligence cytotoxicity assay in co-culture with PBMC or whole cancer biopsy cells (1: 5) and BiTE containing supernatant. Same as above. Effect of immunosuppressed ascites sample on FAP BiTE and anti-CD3 / CD28 bead mediated activity of PBMC T cells. (A) PBMC T cells activated with anti-CD3 / Cd28 dynabeads. (B) PBMC T cells NS activated with control or FAP BITE in the presence of NHDF cells: normal serum, A: peritoneal ascites. FAP BiTE expressing EnAd polarizes the patient's CD11b ⁺ macrophages to a more inflammatory phenotype (A) unpurified total cells from ascites samples in the presence of free BiTE or BiTE expressing virus. Incubated in% ascites. Interferon gamma treatment was used as a positive control. Three days later, the total cell population was collected and the induction of the activation marker CD25 on CD3 ⁺ cells was measured by flow cytometry. (B) The level of interferon gamma in the culture supernatant from (A) was measured by ELISA. (C) On day 3, the expression levels of CD68, CD86, CD206 and CD163 on CD11b + cells from (A) were measured by flow cytometry. Representative flow cytometry spectra from triplicate are shown along with the complete data set. Same as above. Characterization of prostate solid tumor structure and cellular composition (A) EpCAM staining, (B) CD8 staining, (C) FAP staining. (D) Representative immunohistochemical image of CD25 induction in prostate tumor sections after treatment with BiTE expressing virus. Tumor cores were sliced to a thickness of 300 μM with a Leica vibratome, cultured, infected with the insert and harvested after 7 days of treatment. (E) Levels of IFNg in tissue section culture medium measured by ELISA. Supernatants were collected from slice cultures of malignant and benign tissues at specific time points. (F) Levels of IL-2 in tissue culture medium of malignant and benign tissues measured by ELISA. Same as above. Same as above. 77A-C are schematic diagrams of the transgene cassette used in Example 33. FIG. FIG. 77D is a graph showing the number of viral genomes detected per cell in tumor cells treated with NG-611, NG-612 and NG-617. Same as above. After co-culture with EpCAM expressing SKOV cells and recovering supernatant from A549 cells 24, 48 or 72 hours after treatment with NG-611 virus particles, CD69 (a), CD25 (b), HLA-DR (C) Ratio comparing T cells expressing CD40L (d) or cell surface CD107a (e) with NG-612, enadenotucilv or untreated control supernatant. After co-culture with FAP expressing MRC-5 cells and recovering supernatant from A549 cells 24, 48 or 72 hours after treatment with NG-612 virus particles, CD69 (a), CD25 (b), HLA -Ratio of T cells expressing DR (c), CD40L (d) or CD107a (e) on the cell surface to NG-611, enadenochirev or untreated control supernatant. Proportion of MRC-5 cells expressing EpCAM and FAP SKOV cells incubated with supernatant recovered from A549 cells at 24, 48 or 72 hours after treatment with NG-611, NG-612 or enadenotucilv virus particles, or untreated control supernatant IFNγ expression in the supernatant of T cell co-cultures with (A) or MRC-5 cells (B). Anti-tumor efficacy and immune activation of BiTE expressing virus in vivo. (A) Tumor volume in mice treated with saline, enadenotucilv or NG-611. (B) The ratio of CD8 to CD4 T cells in NG-611 treated tumors compared to enadenoticirev treated or untreated controls. Schematic of transgene cassette. (A) NG-615, (b) NG-640, (c) NG-641. Graph showing the number of viral genomes detected per cell in tumor cells treated with NG-612 and NG-615 Expression of IFNα, MIP1α and Flt3 L in the supernatant of NG-615 cell supernatant versus the supernatant of enadenotucirev and untreated control tumor cells. After co-culture with FAP expressing MRC-5 cells and recovering supernatant from A549 cells 24, 48 or 72 hours after treatment with NG-615 virus particles, CD69 (a), CD25 (b), HLA -Numbers comparing T cells expressing DR (c), CD40L (d) or cell surface CD107a (e) with NG-612, endadenocirev or untreated control supernatant. MRC-incubated with supernatant recovered from A549 cells at 24, 48 or 72 hours after treatment with NG-612, NG-615 or enadenotucirev virus particles, or untreated control supernatant IFNγ expression in the supernatant of T cell co-cultures with 5 cells. Schematic diagram of the NG-618 transgene cassette Surface FAP on MRC-5 cells after incubation with supernatant recovered from A549 cells 72 hours after treatment with NG-611, NG-612, NG-615 or enadenotucilv virus particles Expression (a) or (b) detection of EpCAM expression on SKOV cells. After co-culturing with FAP expressing MRC-5 cells and collecting supernatant from A549 cells 72 hours after treatment with NG-618 virus particles, CD24 (a), CD40L (b), or cell surface CD107a ( c) Ratio comparing T cells expressing with an adenotocirev or untreated control. After co-culture with EpCam expressing SKOV cells and recovering supernatant from A549 cells 72 hours after treatment with NG-618 virus particles, CD24 (a), CD40 (b), or cell surface CD107a (c ) Expressing T cells and the ratio of enadenoticirev or untreated controls. Supernatants recovered from T cells and A549 cells were co-cultured 72 hours after treatment with NG-618 virus particles compared to enadenotucilv or untreated controls, followed by dead MRC. -5 (a) or SKOV (b) Percentage of cells.

SEQ ID NO: 1 Anti-EpCAM BiTE DNA coding sequence with N-terminal signal sequence and C-terminal deca-His affinity tag SEQ ID NO: 2 Anti-EpCAM BiTE protein sequence with N-terminal signal sequence and C-terminal deca-His affinity tag 3 Anti-FAP BiTE DNA coding sequence SEQ ID NO: 4 with N-terminal signal sequence and C-terminal deca-His affinity tag Anti-FAP BiTE amino acid sequence SEQ ID NO: 5 N-terminus with N-terminal signal sequence and C-terminal deca-His affinity tag Control (anti-FHA) BiTE DNA coding sequence SEQ ID NO: 6 with signal sequence and C-terminal decaHis affinity tag Control (anti-FHA) BiTE amino acid sequence SEQ ID NO: 6 with N-terminal signal sequence and C-terminal decaHis affinity tag 7 Anti-CD3 S The amino acid sequence of SEQ ID NO: Fv 8 anti-CD3 VH
SEQ ID NO: 9 anti-CD3 VL
SEQ ID NO: 10 Anti-CD3 ScFv linker SEQ ID NO: 11 Anti-FAP ScFv
SEQ ID NO: 12 Anti-FAP VL domain SEQ ID NO: 13 Anti-FAP VH domain SEQ ID NO: 14 Anti-FAP and anti-EpCAM linker sequence SEQ ID NO: 15 BiTE leader sequence SEQ ID NO: 16 Anti-EpCAM ScFv
SEQ ID NO: 17 Anti-EpCAM VL
SEQ ID NO: 18 Anti-EpCAM VH
SEQ ID NO: 19 Control BiTE (anti-FHA)
SEQ ID NO: 20 ScFv of control (anti-FHA)
SEQ ID NO: 21 control (anti-FHA) VL
SEQ ID NO: 22 control (anti-FHA) VH
SEQ ID NO: 23 Control (anti-FHA) ScFv linker sequence SEQ ID NO: 24 Deca-His tag sequence SEQ ID NO: 25 FAP BiTE-P2A-RFP (italics = leader, bold = furin cleavage site, underline = P2A sequence, lower case = RFP)
SEQ ID NO: 26 Control (anti-FHA) BiTE-P2A-RFP (italics = leader, bold = furin cleavage site, underline = P2A sequence, lower case = RFP)
SEQ ID NO: 27 Human EpCAM DNA coding sequence SEQ ID NO: 28 Human EpCAM amino acid sequence SEQ ID NO: 29 Human FAP DNA coding sequence SEQ ID NO: 30 Human FAP amino acid sequence SEQ ID NO: 31 CMV promoter sequence SEQ ID NO: 32 SV40 late polyadenylation sequence SEQ ID NO: 33 Null SEQ ID NO: 34 NG-601 (EnAd-CMV-EpCAMiTE)
Sequence number 35 NG-602 (EnAd-SA-EpCAMBiTE)
SEQ ID NO: 36 NG-605 (EnAd-CMV-FAPBiTE)
Sequence number 37 NG-606 (EnAd-SA-FAPBiTE)
SEQ ID NO: 38 EnAd Genome SEQ ID NO: 39 BX DNA SEQ ID NO: 40 corresponding to and containing EnAd genome base pairs 28166-28366 SEQ ID NO: 40 BY DNA SEQ ID NO: SEQ ID NO: corresponding to EnAd genome base pairs 29345-29379 41 HIS tag SEQ ID NO: 42 Splice acceptor SEQ ID NO: 43 SV40 polyadenylation sequence SEQ ID NO: 44 EpCam BiTE nucleic acid sequence (OKT3)
SEQ ID NO: 45 FAP BiTE nucleic acid sequence (OKT3)
SEQ ID NO: 46 FAP BiTE nucleic acid sequence (aCD3)
SEQ ID NO: 47 NG-611 transgene cassette SEQ ID NO: 48 NG-612 transgene cassette SEQ ID NO: 49 NG-613 transgene cassette SEQ ID NO: 50 restriction site insert (BX)
SEQ ID NO: 51 restriction site insert (BY)
SEQ ID NO: 52 CMV promoter SEQ ID NO: 53 PGK promoter sequence SEQ ID NO: 54 CBA promoter sequence SEQ ID NO: 55 Short splice acceptor (SSA) DNA sequence SEQ ID NO: 56 Splice acceptor (SA) DNA sequence SEQ ID NO: 57 Branched splice acceptor (bSA ) DNA sequence SEQ ID NO: 58 Kozak sequence (null sequence)
SEQ ID NO: 59 Example of start codon SEQ ID NO: 60 Internal ribosome entry sequence (IRES)
SEQ ID NO: 61 P2A peptide SEQ ID NO: 62 F2A peptide SEQ ID NO: 63 E2A peptide SEQ ID NO: 64 T2A peptide SEQ ID NO: 65 polyadenylation (poly A) SEQ ID NO: 66 leader sequence SEQ ID NO: 67 leader sequence SEQ ID NO: 68 IFNΓ amino acid sequence SEQ ID NO: 69 IFNα Amino acid sequence SEQ ID NO: 70 TNFα amino acid sequence SEQ ID NO: 71 DNA sequence corresponding to the E2B region of the EnAd genome (base pairs 10355-5068) SEQ ID NO: 72 Anti-EpCAM BiTE DNA having N-terminal signal sequence and C-terminal decaHis affinity tag Coding sequence SEQ ID NO: 73 with N-terminal signal sequence but without C-terminal decaHis affinity tag, anti-EpCAM BiTE protein SEQ ID NO: 74 with N-terminal signal sequence but C-terminal decaHis parent Anti-FAP BiTE DNA coding sequence SEQ ID NO: 75 with N-terminal signal sequence without sex tag, but without C-terminal decaHis affinity tag, with anti-FAP BiTE amino acid sequence SEQ ID NO: 76 N-terminal signal sequence Has a control (anti-FHA) BiTE DNA coding sequence SEQ ID NO: 77 N-terminal signal sequence but does not have a C-terminal deca-His affinity tag but does not have a C-terminal deca-His affinity tag. FHA) BiTE amino acid sequence SEQ ID NO: 78 Control BITE (anti-FHA) without C-terminal decaHis affinity tag
SEQ ID NO: 79 NG-601 (De-Ad-CMV-EpCAMBiTE) without deca-His affinity tag
SEQ ID NO: 80 NG-602 (EnAd-SA-EpCAMBiTE) without deca-His affinity tag
SEQ ID NO: 81 no NG-605 affinity tag, En-Ad-CMV-FAPBiTE
SEQ ID NO: 82 NG-606 (EnAd-SA-FAPBiTE) without deca-His affinity tag
SEQ ID NO: 83 EpCam BiTE nucleic acid sequence (OKT3)
SEQ ID NO: 84 Null SEQ ID NO: 85 FAP BiTE nucleic acid sequence (OKT3)
SEQ ID NO: 86 Null SEQ ID NO: 87 FAP BiTE nucleic acid sequence (aCD3)
SEQ ID NO: 88 NG-611 transgene cassette SEQ ID NO: 89 NG-612 transgene cassette SEQ ID NO: 90 NG-613 transgene cassette SEQ ID NO: 91 NG-614 transgene cassette SEQ ID NO: 92 NG-617 transgene cassette SEQ ID NO: 93 EpCam BiTE Amino acid sequence (OKT3)
SEQ ID NO: 94 FAP BiTE amino acid sequence (OKT3)
SEQ ID NO: 95 FAP BiTE amino acid sequence (aCD3)
SEQ ID NO: 96 NG-611 Genome SEQ ID NO: 97 NG-612 Genome SEQ ID NO: 98 NG-613 Genome SEQ ID NO: 99 NG-614 Genome SEQ ID NO: 100 NG-617 Genome SEQ ID NO: 101 NG-615 Genome SEQ ID NO: 102 NG-640 Genome Sequence No. 103 NG-641 genome SEQ ID NO: 104 Null sequence SEQ ID NO: 105 Flt3L nucleic acid sequence SEQ ID NO: 106 Null sequence SEQ ID NO: 107 MIP1α nucleic acid sequence SEQ ID NO: 108 flexible linker sequence SEQ ID NO: 109 IFNα nucleic acid sequence SEQ ID NO: 110 CXCL10 nucleic acid sequence SEQ ID NO: 111 CXCL9 Nucleic acid sequence SEQ ID NO: 112 NG-615 transgene cassette SEQ ID NO: 113 NG-640 transgene cassette SEQ ID NO: 114 NG-641 transgene cassette SEQ ID NO: 115 FLT3L Mino acid sequence SEQ ID NO: 116 MIP1α amino acid sequence SEQ ID NO: 117 IFNα amino acid sequence SEQ ID NO: 118 CXCL9 amino acid sequence SEQ ID NO: 119 CXCL10 amino acid sequence SEQ ID NO: 120 NG-618 genome SEQ ID NO: 121 NG-618 EpCam BiTE nucleic acid sequence SEQ ID NO: 122 NG-618 FAP BiTE nucleic acid sequence SEQ ID NO: 123 NG-618 transgene cassette SEQ ID NO: 124-297 Linker sequence SEQ ID NO: 298 NG-616 genome

Example 1
Recombinant BiTE was designed and produced proteins as described in this example.

BiTE engineering BiTE is generated by linking two single chain antibody fragments (ScFv) of different specificities with a flexible Gly ₄ Ser linker. ScFv is generated by linking the _VH and _VL domains from the parent monoclonal antibody with a linker. Each BiTE was designed with an N-terminal signal sequence for mammalian secretion and a C-terminal decahistidine affinity tag for detection and purification. BiTE was manipulated by standard DNA cloning techniques and inserted into a protein expression vector (FIG. 1). Anti-EpCAM BiTE is from patent WO2005040220 (SEQ ID NO: 63 therein) with a signal sequence and an affinity tag added. Anti-FAP BiTE was newly created using anti-FAP ScFv from patent WO20130037835A2 and anti-CD3 ScFv from patent WO2005040220 (SEQ ID NO: 63 therein) with the addition of a signal sequence and an affinity tag. Control BiTE is, Hussein et al., Anti-FHA (Bordetella pertussis from filamentous hemagglutinin) ScFv (Hussein AH et al., 2007, (2007) "Construction and characterization of single-chain variable fragment antibodies directed against the Bordetella pertussis surface adhesins anti-CD3 ScFv from the filamentous hemagglutinin and peractin ". Infect Immunity 75, 5476-5482) and patent WO2005040220 (SEQ ID NO: 63 therein), the signal sequence and It was used in addition to the sum of tag. These BiTE DNA coding sequences and amino acid sequences are SEQ ID NOs: 1-6.

Recombinant BiTE proteins were produced by cloning each sequence into the pSF-CMV vector using the CMV promoter (SEQ ID NO: 31) to drive recombinant BiTE production protein expression (Figure 1). Plasmid DNA concentrations of plasmids, pSF-CMV-EpCAMBiTE, pSF-CMV-FAPBiTE and pSF-CMV-ControlBiTE (Table 2) were measured by NanoDrop. An empty pSF-CMV vector is included as a negative control. Each 54.7 μg was diluted with 4 mL of OptiMEM. 109.2 μg of PEI (linear, MW 25000, Polysciences, USA) was diluted with 4 mL of OptiMEM medium and mixed with 4 ml of diluted DNA to form a DNA-PEI complex (DNA: PEI ratio 1: 2 (w / W)). After incubating at room temperature for 20 minutes, the mixture was replenished to 18 mL with OptiMEM and this transfection mixture was added to a T175 flask containing Ad293 cells at 90% confluence. After incubating the cells with the transfection mixture at 37 ° C., 5% CO ₂ for 4 hours, 30 mL of cell culture medium (glutamine supplemented DMEM high glucose, phenol red free) is added to the cells and the flask is incubated at 37 ° C., 5% CO _2. And incubated for 48 hours. In order to ensure efficient transfection efficiency, another cell flask was transfected with pSF-CMV-GFP in parallel. In order to recover the secreted protein, the supernatant of the transfected cells was recovered and centrifuged at 350 g for 5 minutes at 4 ° C. to remove cellular components (Allegra X-15R, Beckman Coulter). The supernatant was transferred to a 10k MWCO Amicon Ultra-15 centrifugal filter unit (Millipore). After rotating at 4750 rpm and 4 ° C., the volume of retentate was adjusted using a flow-through to obtain a 50-fold higher concentration. An aliquot of the concentrated protein was stored at −80 ° C.
Table 2
A “p” used as a prefix in the naming construct indicates that the construct is a plasmid.

To detect recombinant BiTE detection BiTE, a C-terminal decahistidine affinity tag can be probed with an anti-His antibody using Western blotting techniques. The protein sample was adjusted with lysis buffer to a final volume of 15 μL containing 2.5 μL of 6 × Laemmli SDS sample buffer containing β-mercaptoethanol and SDS. Samples were incubated at 95 ° C. for 5 minutes to denature the protein and loaded onto a 15-well 10% precast polyacrylamide gel (Mini-PROTEAN TGX Precast Gels, BioRad, UK). Gels were run at 180 V for 45 minutes in 1 × running buffer in Mini-PROTEan Tetra System (BioRad, UK). Proteins from the SDS gel were transferred onto nitrocellulose membranes by wet electroblotting for 90 minutes at 300 mA and 4 ° C. in 1 × transcription buffer in Mini Trans-Blot Cell (BioRad, UK). Movement was performed in the presence of an ice pack to limit heat. The nitrocellulose membrane was then blocked with 5% milk in PBS-T on a shaker for 1 hour at room temperature and anti-His (C-terminal) antibody (mouse α-6xHis, clone 3D5, Invitrogen, UK, # 46-0693)) And diluted 1: 5000 with PBS / 5% milk. After incubating overnight at 4 ° C. on a shaker, the membrane was washed and washed with HRP-labeled polyclonal secondary α-mouse-immunoglobulin-antibody (1: 10.000 in PBS / 5% milk, Dako, # P0161). Probed for 1 hour at room temperature. For visualization, SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, UK) was applied according to the manufacturer's instructions, exposed to X-ray film and developed with an automatic film processor. The results showed the expression and secretion of BiTE protein from Ad293 cells transfected with BiTE expression plasmid but not the parent vector.

Quantification of Recombinant BiTE To determine the amount of recombinant BiTE protein, the BiTE signal was converted to a His-tagged (C-terminal 10His) protein standard (10 × His-tagged human cathepsin D, Biolegend using the dot blot technique. , # 556704). BiTE samples and 2-fold serial dilutions of protein standards were prepared and 1.5 μL each was applied directly to a nitrocellulose membrane and air dried for 20 minutes. The blocking and staining protocol described above for Western blotting was then performed. The molar concentration of the protein standard was adjusted to represent a BiTE concentration of 250 μg / mL. The results (FIG. 2A) demonstrated BiTE protein expression and secretion from Ad293 cells transfected with BiTE expression plasmid.

FAP binding ELISA
FAP binding activity of FAP BiTE and control (anti-FHA) BiTE (SEQ ID NOs: 4 and 6) secreted from cells transfected with pSF-CMV-FAPBiTE or pSF-CMV-ControlBiTE are enzyme-linked immunosorbent assays (ELISA) ). An empty pSF-CMV vector supernatant was included as a negative control. Prepare ELISA plates (Nunc Immuno MaxiSorp 96-well microplates) by coating overnight at 4 ° C. with human FAP / seprase protein (100 ng / well, Sino Biological Inc, 10464-H07H-10) in PBS buffer. did. The plate was washed with PBS 0.05% Tween 20 during all subsequent binding steps. Plates were blocked with 5% BSA in PBS 0.05% Tween 20 for 1 hour at room temperature. Proteins taken from wells transfected with a fixed amount of BiTE protein or empty pSF-CMV vector were diluted 10-fold with PBS / 5% BSA / 0.05% Tween 20. All samples were added to the FAP coated plate and incubated for 2 hours at room temperature. Anti-His (C-terminal) antibody (mouse anti-6xHis, clone 3D5, Invitrogen, UK, # 46-0693), a detection antibody, was diluted 1: 1000 and applied for 1 hour at room temperature. HRP-conjugated anti-mouse Fc (1: 1000 in PBS / 5% milk, Dako) was then applied for 1 hour at room temperature, followed by HRP substrate solution 3.3.5.5′-tetramethylethylenediamine (TMB, Thermo-Fisher). ) Was used to perform HRP detection. The reaction was stopped using a stop solution and color development was measured at 450 nm with a plate reader. Absorbance at 450 nm was plotted for FAP BiTE, control BiTE and empty vector supernatant, indicating specific binding of FAP BiTE to FAP protein. The results (FIG. 2B) show specific binding of FAP BiTE but not control BiTE to recombinant FAP protein.

EpCAM binding ELISA
EpCAM binding activity of EpCAM BiTE and control BiTE (SEQ ID NOs: 2 and 6) secreted from cells transfected with pSF-CMV-EpCAMBiTE or pSF-CMV-ControlBiTE were evaluated by enzyme-linked immunosorbent assay (ELISA). . An empty pSF-CMV vector supernatant is included as a negative control. ELISA plates (A Nunc Immuno MaxiSorp 96 well microplates) by coating overnight at 4 ° C. with human EpCAM / TROP-1 protein (50 ng / well, Sino Biological Inc, # 10694-H02H-50) in PBS buffer. ) Was prepared. The plate was washed with PBS 0.05% Tween 20 during all subsequent binding steps. Plates were blocked with 5% BSA in PBS 0.05% Tween 20 for 1 hour at room temperature. Proteins taken from wells transfected with a fixed amount of BiTE protein or empty pSF-CMV vector were diluted 10-fold with PBS / 5% BSA / 0.05% Tween 20. All samples were added to EpCAM coated plates and incubated for 2 hours at room temperature. Anti-His (C-terminal) antibody (mouse anti-6xHis, clone 3D5, Invitrogen, UK, # 46-0693), a detection antibody, was diluted 1: 5000 and applied for 1 hour at room temperature. HRP-conjugated anti-mouse Fc (1: 1000 in PBS / 5% milk, Dako) was then applied for 1 hour at room temperature, followed by HRP substrate solution 3.3.5.5′-tetramethylethylenediamine (TMB, Thermo-Fisher). ) Was used to perform HRP detection. The reaction was stopped using a stop solution and color development was measured at 450 nm with a plate reader. Absorbance at 450 nm was plotted for EpCAM BiTE, control BiTE and empty vector supernatant, demonstrating specific binding of EpCAM BiTE to recombinant EpCAM. The results (FIG. 2C) show specific binding of EpCAM BiTE and no specific binding of control BiTE to recombinant EpCAM protein.

Example 2
Prior to constructing a BiTE transgene-bearing EnAd virus, the functional activity of the recombinant BiTE protein was evaluated in several different assays.

Isolation of human peripheral blood mononuclear cells (PBMC) Density gradient of human PBMC from fresh human blood samples from healthy donors or from whole blood leukocyte cones obtained from NHS Blood and Transplant UK, Oxford Isolated by centrifugation. In each case, the sample was diluted 1: 2 with PBS and 25 mL of this mixture was layered on 13 mL Ficoll (1.079 g / mL, Ficoll-Paque Plus, GE Healthcare) in a 50 mL Falcon tube. Samples were centrifuged at 1600 rpm for 30 minutes at 22 ° C. (Allegra X-15R, Beckman Coulter) at the lowest speed setting to maintain phase separation. After centrifugation, the four layers could be observed to contain a plasma layer on top, followed by a PBMC-containing interface, Ficoll layer, and a red blood cell and granulocyte layer on the bottom. PBMCs were collected using a Pasteur pipette, washed twice with PBS (1200 rpm for 10 minutes at room temperature), and resuspended in RPMI medium supplemented with 10% FBS.

Isolation of CD3 positive T cells CD3 positive (CD3 ⁺⁾ from PBMC by depleting non-CD3 cells using the Pan T cell isolation kit (Miltenyi Biotec, # 130-096-535) according to the manufacturer's protocol. ) T cells were extracted.

Primary Ascites Sample Processing Primary human ascites samples were received from patients with multiple indications including but not limited to ovarian cancer, pancreatic cancer, breast cancer and gastric cancer in the Churchill Hospital (Oxford University Hospital) tumor ward. Upon receipt, cells and liquid fractions were separated in aliquots of fluid frozen at -20 ° C for storage and future analysis. The cell fraction was treated with red blood cell lysis buffer (Roche, # 118143389001) to remove red blood cells according to the manufacturer's instructions. The cell types present in each sample were determined by staining for EpCAM, EGFR, FAP, CD45, CD11b, CD56, CD3, CD4, CD8, PD1 and CTLA4 and analyzed by flow cytometry. The cells were then used fresh for ex vivo T cell activation and target cell lysis experiments. In some cases, cells were passaged in DMEM supplemented with 10% FBS for use in later experiments.

Cell line maintenance As specified in Table 3, all cell lines are 10% (v / v) fetal bovine serum (FBS, Gibco ™) and 1%, unless otherwise noted ( v / v) penicillin / streptomycin (10 mg / mL, Sigma-Aldrich, UK) and humidified incubator (MCO) in DMEM (Sigma-Aldrich, UK) or RPMI medium (Sigma-Aldrich, UK). −17 AIC, Sanyo) at 37 ° C. and 5% CO ₂ . Cells were split every 2-3 days before reaching confluency by enzymatic dissociation with trypsin / EDTA (0.05% trypsin, 0.02% EDTA, Sigma-Aldrich, UK). In this process, media was aspirated and cells were washed with 15 ml PBS followed by treatment of cells with 2 mL trypsin / EDTA for 2-10 minutes at 37 ° C. Trypsin was neutralized with 10 mL DMEM containing 10% FBS and a portion of the cells was transferred to a new flask containing fresh media. For routine cell culture, the media was supplemented with 10% FBS for infection and viral plasmid transfection with 2% FBS and for recombinant BiTE plasmid transfection without FBS.
Table 3

Statistical When two conditions were compared, statistical analysis was performed using a t-test. In all other cases, statistical analysis was performed using one-way analysis of variance.

Characterization of human T cell activation by recombinant FAP BiTE The ability of FAP BiTE to induce T cell activation in the presence or absence of normal human skin fibroblasts (NHDF) was compared. Human CD3 ⁺ T cells (in a 96-well U-bottom plate, 70,000 cells per well) alone or in the presence of 300 ng / mL FAP or control BiTE alone or NHDF cells (10: 1 T: NHDF ). Cells were co-cultured for 24 hours at 37 ° C. and subsequently harvested with enzyme-free cell dissociation buffer (Thermo, # 13151014). The expression levels of CD69 (FIG. 3A) and CD25 (FIG. 3B) on CD45 ⁺ T cells were then analyzed by antibody staining and flow cytometry and expressed as geometric mean fluorescence (gMFI) values. Plate immobilized anti-CD3 antibody (7.5 μg / mL) was used as a positive control for T cell activation. FAP BiTE selectively induced the expression of activation markers CD69 and CD25 on T cells, indicating that it was able to activate T cells.

In a second similar experiment, T cells were cultured 6 hours after co-culture with NHDF cells (200,000 CD3 ⁺ cells ⁺ 40,000 NHDF in wells of 96 well plates) and 300 ng / mL FAP or control BiTE. Evaluation was by intracellular cytokine staining. Brefeldin A was added to the medium 5 hours prior to collection and CD45 ⁺ T cells were intracellularly stained for IFNγ expression. As a positive control, T cells were stimulated with soluble PMA (10 ng / mL) and ionomycin (1 μg / mL). The results shown in FIG. 4A show that FAP BiTE in the presence of NHDF resulted in a significantly higher number of IFNγ expressing T cells compared to control BiTE.

Example 3
A series of experiments similar to those of Example 2 were performed to characterize the recombinant EpCAM BiTE protein.

Characterization of human T cell activation by recombinant EpCAM BiTE The ability of EpCAM BiTE to induce T cell activation in the presence or absence of EpCAM positive DLD cell lines was compared. Human CD3 ⁺ T cells (70,000 cells per well in 96 well U-bottom plate) alone or in the presence of 600 ng / mL EpCAM or control BiTE, alone or DLD cells (10: 1 T: DLD) And co-cultured. Cells were co-cultured at 37 ° C. for 24 hours and then harvested using enzyme-free cell dissociation buffer. The expression levels of CD69 and CD25 on CD45 ⁺ T cells were then analyzed by antibody staining and flow cytometry, and the data were expressed as geometric mean fluorescence (gMFI) values. Plate immobilized anti-CD3 antibody (7.5 μg / mL) was used as a positive control for T cell activation. EpCAM BiTE selectively induced the expression of activation markers CD69 and CD25 on T cells, indicating that it was able to activate T cells (FIGS. 4B and C).

In similar experiments, T cells were treated with DLD cells (200,000 CD3 ⁺ T cells ⁺ 40,000 DLD cells per well of a 96-well plate) and intracellular cytokines 6 hours after co-culture with 300 ng / mL EpCAM or control BiTE. Assessed by staining. Brefeldin A was added to the medium 5 hours prior to collection and CD45 ⁺ T cells were intracellularly stained for IFNγ expression. As a positive control, T cells were stimulated with soluble PMA (10 ng / mL) and ionomycin (1 μg / mL). The results showed that EpCAM BiTE in the presence of DLD resulted in significantly higher numbers of IFNγ expressing T cells compared to control BiTE (FIG. 5A).

In another similar experiment, PBMCs from 8 different blood donors were used to assess donor-dependent variation in BiTE-mediated T cell activation. DLD (7,000 cells) was co-cultured with 100,000 PBMCs in U-bottom 96-well plates in the presence of medium alone or 300 ng / mL control or EpCAM BiTE. Cells were co-cultured at 37 ° C. for 24 hours and subsequently harvested. The expression levels of CD69 and CD25 on CD45 ⁺ T cells were then analyzed by antibody staining and flow cytometry, and the data were expressed as geometric mean fluorescence (gMFI) values. The results showed that EpCAM BiTE induces expression of activation markers CD69 and CD25 in CD3 ⁺ T cells from all 8 donors (FIGS. 5B and C).

Example 4
In this example, the ability of recombinant FAP BiTE activated T cells to induce death of fibroblast target cells was evaluated.

FAP BiTE induces T cell-mediated lysis of FAP positive cell lines and primary cells

  NHDF (7,000 cells) was co-cultured with 70,000 T cells in wells of a U-bottom 96 well plate in the presence of medium alone or 300 ng / mL control or FAP BiTE. After 24 hours of co-culture, the supernatant was collected and cytotoxicity was determined by LDH assay according to the manufacturer's instructions. The results are in FIG. 6A and show that FAP BiTE significantly increased NHDF cell lysis.

In a similar experiment, 7,000 primary lung fibroblasts (BTC100) were co-cultured with 70,000 CD3 ⁺ T cells with or without 300 ng / mL control or FAP BiTE. After 24 hours of co-culture, the supernatant was collected and cytotoxicity was measured by LDH assay. The results in FIGS. 6B and C show that FAP BiTE significantly increased lysis of primary human cancer-related fibroblasts (CAF). The expression of FAP by these and other patient-derived cell lines is shown in FIG.

Co-culture 8,000 NHDF cells with 40,000 T cells at BiTE concentrations ranging from 2 × 10 ³ to 2 × 10 ⁻² ng / mL with FAP BiTE mediated cytolysis. Evaluated by. After co-culture at 37 ° C. for 24 hours, LDH assay was performed on the supernatant to determine target cell cytotoxicity. A dose response curve was fitted using a four parameter non-linear fit model integrated into GraphPad Prism to generate an EC 50 value for 3.2 ng / mL FAP BiTE. The results (FIG. 8A) show a dose-dependent relationship between FAP BiTE concentration and cytotoxicity (shown as Abs ₄₉₀ ) as measured by LDH assay.

Example 5
A study similar to Example 4 was used to evaluate the ability of recombinant EpCAM BiTE activated T cells to induce killing of target tumor cells.

EpCAM BiTE induces T cell-mediated lysis of EpCAM positive cell lines in DLD tumor cells (7,000 cells) in wells of U-bottom 96-well plates in the medium alone or in the presence of 300 ng / mL control or EpCAM BiTE. And co-cultured with 70,000 T cells. After 24 hours of co-culture, the supernatant was collected and cytotoxicity was measured by LDH assay. The results in FIG. 8B show that EpCAM BiTE significantly increased lysis of DLD cells (EpCAM expression on DLD cells is shown in FIG. 8C).

In a similar experiment, 4,000 SKOV cells were co-cultured with 40,000 CD3 ⁺ T cells with or without a 300 ng / mL control or EpCAM BiTE. After 24 hours of co-culture, the supernatant was collected and cytotoxicity was measured by LDH assay. The results in FIG. 9A show that EpCAM BiTE significantly increased lysis of SKOV cells.

In another similar experiment, 5000 MCF7 cells were co-cultured with 50,000 CD3 ⁺ T cells with or without a 300 ng / mL control or EpCAM BiTE. After 24 hours of co-culture, the supernatant was collected and cytotoxicity was measured by LDH assay. The results in FIG. 9B show that EpCAM BiTE also significantly increased lysis of MCF7 cells.

The dose-response relationship for EpCAM BiTE-mediated cell lysis was as follows: 8,000 DLD with 40,000 T cells at EpCAM or control BiTE concentrations ranging from 2 × 10 ³ to 2 × 10 ⁻² ng / mL. Evaluation was made by culturing. After co-culture at 37 ° C. for 24 hours, LDH assay was performed on the supernatant to determine target cell cytotoxicity. A four parameter non-linear fit model integrated into GraphPad Prism was used to fit the dose response curve to generate an EC 50 value for EpCAM BiTE of 7.4 ng / mL. The results in FIG. 10 show a dose-dependent relationship between EpCAM BiTE concentration and cytotoxicity.

  In conclusion, the results of this example demonstrate that EpCAM BiTE was able to induce T cell mediated solubility of multiple EpCAM positive tumor cell lines.

Example 6
Stable CHO and Ad293 cell lines expressing FAP were generated as a means to demonstrate the FAP antigen specificity of FAP BiTE by comparison with parental untransfected cells.

Generation of FAP Expression Stable Transfected Cell Line The protein sequence of the FAP gene was obtained from the NCBI database (SEQ ID NO: 30) and reverse transcribed to generate a DNA coding sequence synthesized by Oxford Genetics Ltd (Oxford, UK). It was. The FAP gene was cloned into the pSF-Lenti vector by standard cloning techniques that produce a pSF-Lenti-FAP vector. HEK293T cells were transfected with a lentiviral FAP expression vector together with pSF-CMV-HIV-Gag-Pol, pSF-CMV-VSV-G, pSF-CMV-HIV-Rev. Lipofectamine 2000 was used as a transfection reagent and added to the vector DNA in a 1: 2 DNA: lipofectamine ratio and incubated with the cells at 37 ° C. The supernatant containing lentivirus was collected after 48 hours and mixed with polybrene (final concentration, 8 μg / mL). Lentivirus / polybrene mixture was added to seeded Ad293 or CHO cells and incubated at 37 ° C. On day 4, the supernatant was replaced with medium containing puromycin (2 μg / mL for Ad293 and 7.5 μg / mL for CHO). Stable mutants were then clonally selected and the FAP expression of the parent cell line or stably transfected mutant was determined by staining with FAP or an isotopic control antibody and analyzed by flow cytometry (FIG. 11A). ).

FAP BiTE-mediated target cell lysis is performed on CHO or CHO-FAP cells (7,000 cells) that are specific for FAP-expressing cells in U-bottom 96-well plates in the medium alone or in the presence of 2 μg / mL control or FAP BiTE. Incubated alone or co-cultured with human T cells (70,000). After 24 hours of incubation, supernatants were collected and target cell cytotoxicity was measured by LDH cytotoxicity assay as described in Example 4 (FIG. 11B). T cell activation was also determined by analyzing the expression levels of CD69 and CD25 by flow cytometry (FIG. 12). Cytotoxicity was only observed when CHO-FAP cells were cultured with T cells and FAP BiTE. This indicates that FAP BiTE mediated T cell activation and target cell lysis is very specific, limited to FAP expressing cells and not a FAP negative parent cell line.

Example 7
Stable CHO and Ad293 cell lines expressing EpCAM were generated as a means to demonstrate EpCAM BiTE EpCAM antigen specificity by comparison with parental non-transfected cells.

Generation of EpCAM expression stable transfected cell line The protein sequence of the EpCAM gene was obtained from the NCBI database (SEQ ID NO: 28) and reverse transcribed to generate a DNA coding sequence synthesized by Oxford Genetics Ltd (Oxford, UK). It was. The EpCAM gene was cloned into the pSF-Lenti vector by standard cloning techniques that produce a pSF-Lenti-EpCAM vector. HEK293T cells were transfected with a lentiviral EpCAM expression vector along with pSF-CMV-HIV-Gag-Pol, pSF-CMV-VSV-G, pSF-CMV-HIV-Rev. Lipofectamine 2000 was used as a transfection reagent and added to the vector DNA in a 1: 2 DNA: lipofectamine ratio and incubated with the cells at 37 ° C. The supernatant containing lentivirus was collected after 48 hours and mixed with polybrene (final concentration, 8 μg / mL). Lentivirus / polybrene mixture was added to seeded Ad293 or CHO cells and incubated at 37 ° C. On day 4, the supernatant was replaced with medium containing puromycin (2 μg / mL for Ad293 and 7.5 μg / mL for CHO). Stable mutants were then clonally selected and EpCAM expression of the parent cell line or stably transfected mutants was determined by staining with EpCAM or an isotopic control antibody and analyzed by flow cytometry (FIG. 13A). ).

EpCAM BiTE-mediated target cell lysis is performed on CHO or CHO-EpCAM cells (7,000 cells) that are specific for EpCAM-expressing cells in U-bottomed 96-well plates in the medium alone or in the presence of 2 μg / mL control or EpCAM BiTE. Incubated alone or co-cultured with human T cells (70,000). After 24 hours of incubation, supernatants were collected and target cell cytotoxicity was measured by LDH cytotoxicity assay (FIG. 13B). T cell activation was also determined by analyzing CD69 and CD25 expression levels by flow cytometry (FIG. 14). Cytotoxicity was only observed when CHO-EpCAM cells were cultured with T cells and EpCAM BiTE. This indicates that EpCAM BiTE-mediated T cell activation and target cell lysis is highly specific, limited to EpCAM-expressing cells and not an EpCAM-negative parent cell line.

Example 8
In further experiments, the ability of recombinant FAP BiTE protein to activate CD4 or CD8 T cells and the ability of each of these T cell subsets to lyse NHDF cells was evaluated. CD3 ⁺ T cells (35,000) were co-cultured with 7,000 NHDF cells in the presence of 300 ng / mL control or FAP BiTE in the wells of a U-bottom 96-well plate and 24 hours at 37 ° C. Incubated for hours. Cells were harvested, stained with antibodies for CD4 or CD8 and CD69 and CD25 and analyzed by flow cytometry. The results (FIG. 15A) demonstrated that FAP BiTE induces increases in activation markers CD69 and CD25 in both CD4 ⁺ and CD8 ⁺ T cells.

In a similar experiment, the ability of each T cell subset (CD4 and CD8) to kill target cells was evaluated. Extract CD4 ⁺ T cells from CD3 purified cells by positive selection using CD4 T cell isolation kit (Miltenyi Biotec, # 130-045-101) with CD8 cells in non-isolated flow-through according to manufacturer's protocol did. In a well of a U-bottom 96-well plate, 7,000 NHDFs were co-cultured with 35,000 CD4 ⁺ or CD8 ⁺ T cells with 300 ng / mL control or FAP BiTE and incubated at 37 ° C. . After 24 hours, the supernatant was collected and the cytotoxicity of the target cells was measured by LDH cytotoxicity assay. The results (FIG. 15B) show that FAP BiTE induces both CD4 ⁺ and CD8 ⁺ T cells to kill NHDF cells.

Example 9
The ability of EpCAM BiTE to activate CD4 ⁺ or CD8 ⁺ T cells and to lyse each subset of DLD tumor cells was evaluated. CD3 ⁺ T cells (35,000) were co-cultured with 7,000 DLD cells in the presence of 300 ng / mL control or EpCAM BiTE in wells of a U-bottom 96-well plate at 24 ° C. Incubated for hours. Cells were harvested, stained with antibodies for CD4 or CD8 and CD69 and CD25 and analyzed by flow cytometry. The results (FIG. 16A) demonstrated that EpCAM BiTE induced an increase in activation markers CD69 and CD25 in both CD4 ⁺ and CD8 ⁺ T cells.

In a similar experiment, the ability of each T cell subset (CD4 and CD8) to kill target cells was evaluated. CD4 ⁺ T cells were extracted from CD3 purified cells by positive selection with CD8 cells in the unselected flow-through using the CD4 T cell isolation kit according to the manufacturer's protocol. In wells of a U-bottom 96-well plate, 7,000 DLDs were co-cultured with 35,000 CD4 ⁺ or CD8 ⁺ T cells with 300 ng / mL control or EpCAM BiTE and incubated at 37 ° C. After 24 hours, the supernatant was collected and the cytotoxicity of the target cells was measured by LDH cytotoxicity assay (FIG. 16B). The results show that EpCAM BiTE induces both CD4 ⁺ and CD8 ⁺ T cells to kill DLD cells.

Example 10
Characterization of FAP BiTE-mediated activation of autologous tumor-related lymphocytes from primary malignant ascites To assess BiTE protein activity using cells from cancer patients, including both CD3 ⁺ T cells and FAP ⁺ cells A sample of primary malignant ascites was obtained for testing. Unpurified ascites cells (thus unchanged from the time of receipt) are placed in U-bottom 96-well plates in either 100% ascites or medium supplemented with 1% human serum in the presence of 500 ng / mL control or FAP BiTE. Seed at 250,000 cells per well. Untreated wells were used as negative controls. After 5 days incubation at 37 ° C., the total cell population was collected and the number of CD3 ⁺ T cells (FIG. 17A) and the expression level of CD25 on CD3 ⁺ T cells was determined (FIG. 17B). The total number of cells per well was determined using precision counting beads. The results demonstrate that FAP BiTE resulted in a significant increase in T cell activation of tumor associated T cells from cancer patients.

As an extension of the experiment, duplicate wells were collected and the number of FAP ⁺ cells was determined by flow cytometry (FIG. 17C). The total number of cells per well was determined using precision counting beads. The results show that FAP BiTE resulted in a significant reduction in the number of self-FAP expressing cells in the ascites sample.

Example 11
Recombinant BiTE-expressing EnAd virus was engineered, produced and purified using the methods described below.

Generation of BiTE-expressed Enadenotucilv EnAd contains frequent heterologous nucleotide substitutions of Ad3 for Ad11p in the E2B region, almost complete E3 deletions, and smaller E4 deletions mapped to E4orf4 , A replication-competent chimeric group B adenovirus (Kuhn et al., Directed evolution generations of novel viral for the treatment of colon cancer, PLoS One, June 18, 2008; e. A schematic of the adenovirus genome used in this study is shown in FIG. 18A.

Using plasmid pEnAd2.4, plasmid pEnAd2.4-CMV-EpCAMBiTE, by direct insertion of a cassette encoding EpCAM BiTE (SEQ ID NO: 1), FAP BiTE (SEQ ID NO: 3) or control BiTE (SEQ ID NO: 5), pEnAd2.4-SA-EpCAMBiTE, pEnAd2.4-CMV-FAPBiTE, pEnAd2.4-SA-FAPBiTE, pEnAd2.4-CMV-Control BiTE, pEnAd2.4-SA-Control BiTE (Table 4) were generated. The transgene cassette is 5 'short splice acceptor sequence (SEQ ID NO: 33) or exogenous CMV promoter (SEQ ID NO: 31), EpCAM, FAP or control BiTE cDNA sequence, and 3' polyadenylation sequence (SEQ ID NO: 32). Was included. Plasmid construction was confirmed by DNA sequencing. The exogenous CMV promoter is constitutively active, thus leading to early expression of the transgene. The splice acceptor sequence drives expression under the control of the viral major late promoter, resulting in subsequent transgene expression after initiation of viral genome replication. The kinetics of this promoter driven expression can be observed in FIG. 18B, where GFP was used as the transgene.
Table 4

Virus production and characterization Plasmids EnAd2.4-CMV-EpCAMBiTE, pEnAd2.4-SA-EpCAMBiTE, pEnAd2.4-CMV-FAPBiTE, pEnAd2.4-SA-FAPBiTE, pEnAd2.4-CMV-Control BiTE, pEnAd2.4. -SA-control BiTE was linearized by restriction digestion with the enzyme AscI to generate a liner virus genome. The digested DNA was purified by isopropanol extraction and precipitated at −20 ° C. for 16 hours in 300 μl> 95% molecular biology grade ethanol and 10 μl 3M sodium acetate. The precipitated DNA was pelleted by centrifuging at 14000 rpm for 5 minutes, washed with 500 μl of 70% ethanol, and then centrifuged again at 14000 rpm for 5 minutes. Clean DNA pellets were air dried and resuspended in 100 μL water. 6.25 μg of DNA was mixed with 15.6 μL of Lipofectamine transfection reagent in OptiMEM and incubated for 20 minutes at room temperature. The transfection mixture was then added to a T-25 flask containing Ad293 cells grown to 80% confluency. After incubating the cells with the transfection mixture at 37 ° C. for 4 hours, 4 ml of 5% CO ₂ cell culture medium (DMEM high glucose with glutamine supplemented with 10% FBS) was added to the cells and the flask was incubated at 37 ° C., 5% CO 2. Incubated at ₂ . Transfected Ad293 cells were monitored every 24 hours and supplemented with additional media every 48-72 hours. Virus production was monitored by observation of significant cytopathic effect (CPE) in the cell monolayer. Once extensive CPE was observed, virus was harvested from Ad293 cells by three freeze-thaw cycles. Single virus clones were selected by serial dilution of the collected lysates, reinfection with Ad293 cells, and collection of wells containing a single plaque. To amplify the virus stock, Ad293 cells were serially infected when the infection reached full CPE. Viable virus production during amplification was confirmed by observation of significant CPE in the cell monolayer.

Virus Purification Once a strong virus stock is amplified, the virus is purified by double cesium chloride density gradient centrifugation (banding) to obtain NG-601, NG-602, NG-603, NG-604, NG-605 and NG- A 606 virus stock was generated. These stocks were titrated by micoBCA assay (Life Technologies) according to manufacturer's instructions (Table 5).
Table 5

Example 12
The activities of NG-601, NG-602, NG-603, NG-604, NG-605 and NG-606 viruses were characterized using the following method.

Characterization of BiTE encoding EnAd activity compared to EnAd in cancer cell lines The ability of NG-601, NG-602, NG-603, NG-604, NG-605, NG-606 or EnAd to replicate A549 lung cancer Analyzed by infection of cells and assessed by qPCR. A549 cells were seeded in wells of a 24-well plate at a cell density of 2 × 10 ⁵ cells / well. Plates were incubated at 37 ° C., 5% CO ₂ for 18 hours before cells were infected with 100 virus particles per cell (ppc) or left uninfected. Wells were harvested 24, 48 or 72 hours after infection and DNA was purified using the PureLink Genomic DNA Mini Kit (Invitrogen) according to the manufacturer's protocol. The total viral genome was quantified by qPCR with each extracted sample or standard using the EnAd hexon gene specific primer-probe set in the reaction mixture detailed in Table 6. qPCR was performed according to the program in Table 7.
Table 6

Table 7

Quantification of the number of detected viral genomes per cell is similar to NG-601, NG-602, NG-603, NG-604, NG-605, NG-606 and EnAd virus replication in the A549 cell line. It was proved that there was (FIG. 19A).

The oncolytic activity of NG-601, NG-602, NG-603, NG-604, NG-605, NG-606 or EnAd was assessed by A549 infection (FIG. 19B). A549 cells were seeded in 96 well plates at a cell density of 1.5 × 10 ⁴ cells / well. Cells were incubated 18 hours at 37 ° C., 5% CO ₂ before cells were infected or left uninfected with increasing ppc of virus (5 × serial dilution, 4.1 × 10 ^{−7 to} 5000 virus ppc). Incubated with. On day 5, A549 cytotoxicity was measured by CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) (Promega, # G3582). Dose response curves were fitted using a four parameter non-linear fit model integrated into GraphPad Prism. The IC50 values generated for each virus demonstrate that the oncolytic activity of NG-601, NG-602, NG-603, NG-604, NG-605, NG-606 and EnAd is comparable for each virus. did.

Confirmation of functional BiTE transgene expression from NG-601, NG-602, NG-603, NG-604, NG-605, NG-606 Viruses NG-601, NG-602, NG-605, NG-606 To determine whether to produce functional BiTE, a T cell activation assay was performed using CHO, CHO-EpCAM and CHO-FAP cell lines as target cells. 10,000 target cells were co-cultured with 50,000 CD3 ⁺ T cells in wells of a U-bottom 96-well plate containing Ad293 virus supernatant diluted 100-fold in culture, Incubated with% CO ₂ for 24 hours. T cells were harvested, stained with antibodies specific for CD25 and CD69, and analyzed by flow cytometry. The results (FIGS. 20A and 20B) show that viruses NG-601 and NG-602 express functional BiTE transgenes that activate T cells when co-cultured with CHO-EpCAM cells, while NG-605 and NG-606 It was shown that T cells were activated when co-cultured with CHO-FAP cells, but expressed a functional BiTE transgene that was not activated when co-cultured with CHO cells.

Quantification of BiTE expression in colon cancer cell lines The amount of BiTE expression by NG-601, NG-602, NG-605, NG-606 infection of human colon cancer cell line DLD was evaluated. DLD cells were seeded in 6-well culture plates at a density of 1.2 × 10 ⁶ cells per well. 18 hours after seeding, DLD cells were infected with EnAd, NG-601, NG-602, NG-603, NG-604, NG-605, NG-606 at 100 ppc. Cells were cultured for 72 hours before the supernatant was recovered from the wells and centrifuged at 1200 rpm for 5 minutes to remove cell debris. The clarified supernatant was then used for killing assays using cytotoxicity compared to a standard curve generated using known concentrations of recombinant BiTE to determine the amount of BiTE in the viral supernatant.

To determine the amount of FAP BiTE produced from NG-605 and NG-606, 8,000 NHDFs, 40,000 CD3 ⁺ T cells and 1 in 10 ³ , 1 in 10 ⁴ , and 10 Cytotoxicity assays were performed by co-culturing with DLD virus supernatant diluted at a ratio of 1 to ⁵ . Standard curves were generated by incubating NHDF and CD3 ⁺ T cells with FAP or control BiTE at 10-fold serial dilutions from 3333 to 3.33 × 10 ⁻⁴ ng / μL. Supernatants were collected 24 hours after treatment and cytotoxicity was measured by LDH assay. The amount of BiTE expressed was determined by comparing the cytotoxicity of the viral supernatant with that of the recombinant BiTE standard curve. The results (FIG. 21) showed that viruses NG-605 and NG-606 produced 9.8 and 49.2 μg of FAP BiTE per million DLD cells, respectively.

From NG-601 and NG-602 to determine the amount of EpCAM BiTEs produced, 8,000 DLD cells, 40,000 CD3 ⁺ T cells and 10 ³ to 1, 10 ⁴ 1, And cytotoxicity assays were performed in co-culture with DLD virus supernatant diluted 1 to 10 ⁵ . Standard curves were generated by incubating DLD and CD3 ⁺ T cells with EpCAM or control BiTE at 10-fold serial dilutions from 3333 to 3.33 × 10 ⁻⁴ ng / μL. Supernatants were collected 24 hours after treatment and cytotoxicity was measured by LDH assay (Figure 22). The amount of BiTE expressed was determined by comparing the cytotoxicity of the viral supernatant with that of the recombinant BiTE standard curve. The results showed that viruses NG-601 and NG-602 produced 165 and 50.3 μg EpCAM BiTE per million DLD cells, respectively.

Example 13
In addition to encoding FAP or control BiTE, NG-607, NG-608, NG-609, NG-610 viruses were also detected in infected cells using fluorescence microscopy (SEQ ID NOs: 25 and 26, Table 4). It carries a red fluorescent protein (RFP) transgene for visualization. The functional activity of these viruses was characterized using the following method.

Confirmation of transgene expression from NG-607, NG-608, NG-609, NG-610 The ability of viruses NG-607, NG-608, NG-609 and NG-610 to produce their BiTE transgenes Ad293 Assessed by cell infection. Ad293 cells were plated into 6-well plates at 1 × 10 ⁶ cells / well. Plates were incubated for 24 hours at 37 ° C., 5% CO ₂ before cells were infected with virus at 100 ppc or left uninfected. Forty-eight hours after infection, plaques were irradiated with a fluorescent mercury lamp and photographed (FIG. 23). The results suggested that viruses NG-607, NG-608, NG-609 and NG-610 expressed the RFP transgene.

Example 14
In the next series of experiments, EnAd and FAP, or control BiTE viruses NG-603, NG-604, NG-605, NG-606, NG-607, NG-608, NG-609, NG-610, tumor cells And the ability to kill target cells including fibroblasts.

In the first study, the ability of EnAd to kill DLD cells was evaluated using the xCELLligence technique. DLD cells were plated at 1.2 × 10 ⁴ cells / well in 48 well E plates and cells were infected with 100 EnAd ppc or left uninfected for 18 hours at 37 ° C., 5% CO ₂ . Incubated for hours. XCELLligence was used to measure target cell cytotoxicity every 15 minutes over an 8-day incubation period. The results (FIG. 24A) suggest that EnAd was able to effectively kill DLD cells over that period.

In a similar experiment, the ability of EnAd to kill SKOV cells was evaluated using the xCELLligence technique. SKOV cells are plated into 48-well E plates at 1 × 10 ⁴ cells / well and 18 hours at 37 ° C., 5% CO ₂ before cells are infected with 100 EnAd ppc or left uninfected. Incubated. Target cell cytotoxicity was measured every 15 minutes for 8 days using xCELLligence. The results (FIG. 24B) suggest that SKOV cells are resistant to EnAd-mediated cytotoxicity over this period.

In a similar experiment, the ability of EnAd to kill NHDF cells was also evaluated using xCELLligence technology. NHDF cells are plated at 4 × 10 ³ cells / well in 48 well E plates and incubated for 18 hours at 37 ° C., 5% CO ₂ before cells are infected or left uninfected with 100 EnAd ppc. did. Using xCELLligence, target cell cytotoxicity was measured every 15 minutes over the same period as A549 and SKOV cells. The results (FIG. 24C) suggest that EnAd is unable to kill NHDF cells within the observed period.

In a similar experiment, the ability of NG-603, NG-604, NG-605, NG-606, and EnAd to kill NHDF cells was evaluated in co-culture with SKOV tumor cells and CD3 ⁺ T cells using xCELLligence. . NHDF cells and SKOV cells were seeded in 48-well E-plates at 4 × 10 ³ and 1 × 10 ³ cells / well, respectively. Plates were incubated for 18 hours at 37 ° C., 5% CO ₂ before cells were infected or left uninfected with 100 ppc EnAd, NG-603, NG-604, NG-605 or NG-606. . After 2 hours of incubation, 37,500 CD3 ⁺ T cells were added to each well. Target cell cytotoxicity was measured every 15 minutes using xCELLligence. The results (FIG. 25A) show that the FAP BiTE expressing viruses NG-605 and NG606 depend on the promoter used for BiTE expression (the CMV promoter is faster) rather than the EnAd or control BiTE expressing viruses NG-603 and NG-604. This demonstrates that lysis of NHDF cells could be induced.

In a similar experiment, the ability of NG-603, NG-604, NG-605, NG-606 and EnAd to kill NHDF cells was demonstrated in co-culture with SKOV and CD3 ⁺ T cells using an LDH cytotoxicity assay. evaluated. NHDF cells and SKOV cells were seeded in 96-well U-bottom plates at 8 × 10 ³ and 2 × 10 ³ cells / well, respectively, and 100 ppc of EnAd, NG-603, NG-604, NG-605 or NG-606. Either infected or left uninfected. After 2 hours of incubation, 75,000 CD3 ⁺ T cells were added to each well and the plates were incubated at 37 ° C., 5% CO ₂ . Supernatants were collected at 0, 24, 48 and 96 hours after treatment and cytotoxicity was measured by LDH cytotoxicity assay. The results (FIG. 25B) show that FAP BiTE-expressing viruses NG-605 and NG606, but not EnAd or control BiTE-expressing viruses NG-603 and NG-604, lysed NHDF cells with promoter-dependent kinetics used for BiTE expression. It is proved that it was able to guide.

  As an extension of the LDH experiment described above, cells were also collected at 0, 24, 48 and 96 hours after treatment, stained with antibodies for CD45, CD69 and CD25 and analyzed by flow cytometry. The results (FIG. 26) show that FAP BiTE-expressing viruses NG-605 and NG-606, but not EnAd or control BiTE-expressing viruses NG-603 and NG-604, are kinetically dependent on the promoter used for BiTE expression. Demonstrate that cell activation could be induced.

In a similar experiment, the dependence on FAP to induce FAP BiTE-mediated T cell activation was evaluated. In 96-well U-bottom plates, SKOV cells were seeded at 2 × 10 ³ cells / well alone or in combination with NHDF cells at 8 × 10 ³ cells / well. Viral particles were added to each well at 100 ppc and the plates were incubated at 37 ° C., 5% CO ₂ . After 2 hours, 75,000 CD3 ⁺ T cells were added and the plates were further incubated. Cells were harvested 96 hours after infection, stained for CD45 and CD25, and analyzed by flow cytometry (FIG. 27A). The results demonstrate that FAP BiTE expressing viruses NG-605 and NG-606 induced T cell activation only in the presence of FAP positive NHDF cells.

In a similar experiment, the specificity of promoter (CMV or viral MLP / SA) driven BiTE expression in NG-605 and NG-606 was further investigated. NHDF cells were seeded at 4 × 10 ³ cells / well in a 96-well U-bottom plate. 100 virus particles per cell were added to each well and the plates were incubated at 37 ° C., 5% CO ₂ . After 2 hours, 40,000 CD3 cells were added and the plates were further incubated. Supernatants were collected 72 hours after infection and cytotoxicity was measured by LDH cytotoxicity assay. The results (FIG. 27B) show that CMV-driven virus NG-605 was able to mediate killing of NHDF cells upon infection with NHDF cells alone, but not SA-driven NG-606. Indicates.

  The results show that both NG-605 and NG-606 were able to induce T cell activation and target cell lysis, but the kinetic profile was slightly different depending on the promoter used.

Time-lapse videos were obtained to observe the viral or T cell mediated lysis of target cells with recombinant FAP BiTE, EnAd, NG-603 or NG-605. NHDF cells were stained with CellTracker Orange CMTMR dye (LifeTech, # C2927), and CD3 ⁺ T cells were stained with CellTrace Violet Cell Proliferation Kit (LifeTech, # C34557) according to the manufacturer's protocol. Stained NHDF was co-cultured with 1.35 × 10 ⁴ DLD or SKOV tumor cells and plated into 24-well plates at 7.5 × 10 ³ cells / well. Plates were incubated for 18 hours at 37 ° C., 5% CO ₂ . Cells were then treated with 300 ng / mL FAP BiTE, infected with 100 ppc EnAd, NG-603, and NG-605, or left untreated. After 2 hours of incubation, 100,000 stained CD3 ⁺ T cells were added to the required wells in addition to 1.5 μM CellEvent Caspase 3-7 reagent (LifeTech, # C10423). Videos were taken with a Nikon TE 2000-E Eclipse inverted microscope and taken every 15 minutes for 96 hours. A video frame is shown in FIG. The results indicate that recombinant FAP BiTE and NG-605 but not EnAd or NG-603 were able to induce rapid lysis of NHDF cells.

In similar experiments, NHDF cells were stained with CellTracker Green CMFDA dye (LifeTech, # C2925) and CD3 ⁺ T cells were stained with CellTrace Violet Cell Proliferation Kit (LifeTech, # C34557) according to the manufacturer's protocol. Stained NHDF was co-cultured with 1.35 × 10 ⁴ DLD or SKOV tumor cells and plated into 24-well plates at 7.5 × 10 ³ cells / well. Plates were incubated for 18 hours at 37 ° C., 5% CO ₂ . Cells were then infected with 100 ppc NG-607, NG-608, NG-609 or NG-610 or left uninfected. After 2 hours of incubation, 100,000 stained CD3 ⁺ T cells were added to the required wells. Videos were taken with a Nikon TE 2000-E Eclipse inverted microscope and taken every 15 minutes for 96 hours. A video frame is shown in FIG. The results show that all viruses resulted in tumor cell infection (RFP, red fluorescence, positive), but only NG-609 and NG-610 were able to induce rapid lysis of co-cultured NHDF cells.

Example 15
In this series of experiments, the ability of EnAd and EpCAM or control BiTE viruses NG-601, NG-602, NG-603 and NG-604 to kill target cells including tumor cells and fibroblasts was evaluated.

Characterization of human T cell activation and EpCAM positive target cell lysis by EnAd, NG-601, NG-602, NG-603 and NG-604. EnAd and NG-601 in the presence or absence of CD3 ⁺ T cells. , NG-602, NG-603 and NG-604 were assessed for their ability to kill DLD tumor cells using xCELLligence technology. DLD cells were plated on 48-well E plates at 1.2 × 10 ⁴ cells / well. Plates were incubated for 18 hours at 37 ° C., 5% CO ₂ before cells were infected with EnAd at 100 ppc or left uninfected. Two hours after infection, 75,000 CD3 ⁺ T cells were added to the required wells. The cytotoxicity of the target cells was measured every 15 minutes using XCELLligence. The results (FIG. 30) show that NG-601 and NG-602 result in significantly faster DLD cytotoxicity in a T cell dependent manner.

In a similar experiment, the ability of EnAd and NG-601, NG-602, NG-603, and NG-604 to kill DLD tumor cells in the presence or absence of CD3 ⁺ T cells was determined by an LDH cytotoxicity assay. Was used to evaluate. DLD cells were plated in 96 well U-bottom plates at 2 × 10 ⁴ cells / well and infected with 100 ppc EnAd or left uninfected. Two hours after infection, 150,000 CD3 ⁺ T cells were added to the required wells. Plates were incubated at 37 ° C., 5% CO ₂ and supernatants were collected and analyzed by LDH cytotoxicity assay at 0, 24, 48 and 72 hours after infection. The results (FIG. 31) show that NG-601 and NG-602 cause more rapid DLD cytotoxicity in a T cell-dependent manner.

As an extension of the LDH experiment described above, cells were also collected at 0, 24, 48 and 96 hours after treatment, stained with antibodies for CD45, CD69 and CD25, and analyzed by flow cytometry to determine the activity of CD3 ⁺ T cells. The crystallization state was determined. The results (FIG. 32) show that EpCAM BiTE-expressing viruses NG-601 and NG-602, but not EnAd or control BiTE-expressing viruses NG-603 and NG-604, are kinetically dependent on the promoter used for BiTE expression. It shows that cell activation could be induced.

In another experiment, the ability of NG-601 to kill DLD tumor cells at various multiplicity of infection (MOI) in the presence or absence of CD3 ⁺ T cells was assessed using the xCELLligence technique. DLD cells were plated on 48 well E plates at 2 × 10 ⁴ cells / well. Cells were incubated for 18 hours at 37 ° C., 5% CO ₂ before cells were infected with NG-601 with MOI (ppc) varying from 0.001 to 10 or left uninfected. Two hours after infection, 150,000 CD3 ⁺ T cells were added to the required wells. Target cell cytotoxicity was measured every 15 minutes using xCELLligence. The results (FIG. 33) show that NG-601 leads to more rapid DLD cytotoxicity in a T cell dependent manner with a MOI as low as 0.001.

In a similar experiment, the ability of EnAd and NG-601, NG-602, NG-603, and NG-604 to kill SKOV tumor cells in the presence or absence of CD3 ⁺ T cells using xCELLligence technology. evaluated. SKOV cells were plated at 1 × 10 ⁴ cells / well on 48 well E plates. Plates were incubated for 18 hours at 37 ° C., 5% CO ₂ before cells were infected or left uninfected with EnAd (100 ppc). Two hours after infection, 50,000 CD3 ⁺ T cells were added to the required wells. Target cell cytotoxicity was measured every 15 minutes using xCELLligence. The results (FIG. 34) suggest that SKOV cells are resistant to EnAd-mediated cytotoxicity over the duration of this study, whereas NG-601 and NG-602 are in the presence of CD3 ⁺ T cells. Was able to induce rapid lysis of SKOV cells.

In a similar experiment, the ability of EnAd and NG-601, NG-602, NG-603 and NG-604 to kill SKOV cells in the presence or absence of CD3 ⁺ T cells was tested using the LDH cytotoxicity assay. And evaluated. SKOV cells were plated in 96 well U-bottom plates at 2 × 10 ⁴ cells / well and infected with EnAd (100 ppc) or left uninfected. Two hours after infection, 150,000 CD3 ⁺ T cells were added to the required wells. Plates were incubated at 37 ° C., 5% CO ₂ and supernatants were collected and analyzed by LDH cytotoxicity assay at 0, 24, 48 and 72 hours after infection. The results (Figure 35) are consistent with previous data, suggesting that SKOV cells are resistant to EnAd-mediated cytotoxicity over this time frame, whereas NG-601 and NG-602 are CD3 ⁺ In the presence of T cells, it was possible to induce rapid lysis of SKOV cells.

As an extension of the LDH experiment described above, cells were also collected at 0, 24, 48 and 96 hours after treatment, stained with antibodies for CD45, CD69 and CD25 and analyzed by flow cytometry to determine the activity of CD3 ⁺ T cells. The activation state was determined (FIG. 36). The results show that EpCAM BiTE-expressing viruses NG-601 and NG-602, but not EnAd or control BiTE-expressing viruses NG-603 and NG-604, activated T cells with kinetics dependent on the promoter used for BiTE expression. Indicates that it was able to be guided.

In a similar experiment, the ability of EnAd and NG-601, NG-602, NG-603, and NG-604 to activate CD3 ⁺ T cells from cancer patients from CD3 ⁺ EpCAM negative primary ascites samples was evaluated. . EpCAM positive DLD cells were plated at 1 × 10 ⁴ cells per well in 96 well U bottom plates and co-cultured with 100,000 ascites cells (as received). Cells were infected with virus particles at 100 ppc or left uninfected. After incubating at 37 ° C. for 48 hours, the total cell population was collected and the expression level of CD25 on CD3 ⁺ T cells was determined by flow cytometry. The results (FIG. 37) show that EpCAM BiTE expressing viruses NG-601 and NG-602, but not EnAd or control BiTE expressing viruses NG-603 and NG-604, induced T cell activation of patient-derived CD3 ⁺ T cells. Show that you can.

  The results showed that both EpCAM BiTE viruses NG-601 and NG-602 were able to induce T cell activation and target cell lysis, but the kinetic profile was slightly different depending on the promoter used. .

Time-lapse videos were obtained and observed for viral or T cell-mediated lysis of target cells by recombinant EpCAM BiTE, EnAd, NG-601 or NG-603. NHDF cells were stained with CellTracker Orange CMTMR dye (LifeTech, # C2927), and CD3 ⁺ T cells were stained with CellTrace Violet Cell Proliferation Kit (LifeTech, # C34557) according to the manufacturer's protocol. Stained NHDF was co-cultured with 1.35 × 10 ⁴ DLD or SKOV tumor cells and plated into 24-well plates at 7.5 × 10 ³ cells / well. Plates were incubated for 18 hours at 37 ° C., 5% CO ₂ . Cells were then treated with 300 ng / mL EpCAM BiTE, infected with EnAd, NG-601 or NG-603 at 100 ppc, or left untreated. After 2 hours of incubation, 100,000 stained CD3 ⁺ T cells were added to the required wells in addition to 1.5 μM CellEvent Caspase 3-7 reagent (LifeTech, # C10423). Videos were taken with a Nikon TE 2000-E Eclipse inverted microscope and taken every 15 minutes for 96 hours. A video frame is shown in FIG. The results show that recombinant EpCAM BiTE and NG-605 result in rapid lysis of both DLD and SKOV target cells, but NHDF remains unaffected.

Example 16
In this example, the activation of FAP ⁺ primary malignant ascites-derived autologous tumor-related lymphocytes from cancer patients by EnAd, NG-603, NG-604, NG-605 and NG-606 was evaluated. Patient samples considered suitable for further analysis included CD3 ⁺ T cells and FAP ⁺ cells.

In the first experiment, unpurified ascites cells obtained from the patient (and therefore as received) were seeded in 2% cells per well of a U-bottom 96-well plate in 100% ascites. Cells were infected with virus at 100 ppc and untreated wells were used as negative controls. EnAd-CMV-GFP and EnAd-SA-GFP were also included in the experiment as reporters to determine infection and late viral gene expression, respectively, and a photomicrograph is shown in FIG. After 5 days incubation at 37 ° C., the total cell population was collected and the expression level of CD25 on CD3 ⁺ T cells (FIG. 40A) was determined. The total number of cells per well was determined using precision counting beads. The results demonstrate that FAP BiTE viruses NG-605 and NG-606 resulted in a significant increase in T cell activation of tumor associated lymphocytes.

As an extension of the experiment, duplicate wells were collected and the number of endogenous FAP ⁺ cells was determined by flow cytometry. The total number of cells per well was determined using precision counting beads. The results (FIG. 40B) show that NG-605 and NG-606 in the ascites sample resulted in a significant decrease in the number of self-FAP expressing cells, which caused some FAP ⁺ cells to be killed by activated T cells. It is suggested that it was made.

In a second experiment, unpurified ascites cells from a cancer patient (and thus unchanged) were placed in either U-bottom 96-well plates in either 100% ascites or medium supplemented with 1% human serum. Of 250,000 cells per well. Cells were infected with virus at 100 ppc and untreated wells were used as negative controls. EnAd-CMV-GFP and EnAd-SA-GFP are also included as reporters to determine infection and late viral gene expression, respectively, and the photomicrograph is shown in FIG. After 5 days incubation at 37 ° C., the total cell population was collected and the number of CD3 ⁺ T cells (FIG. 42) and the expression level of CD25 on CD3 ⁺ T cells (FIG. 43) were determined. The total number of cells per well was determined using precision counting beads. The results demonstrate that, for this patient, recombinant FAP BiTE and NG-605 but not NG-606 resulted in a significant increase in T cell activation of tumor-associated lymphocytes in the medium. Neither virus caused activation in ascites.

As an extension of the experiment, duplicate wells were collected and the number of FAP ⁺ cells was determined by flow cytometry (FIG. 44). The total number of cells per well was determined using precision counting beads. The results show that recombinant FAP BiTE and NG-605 but not NG-606 resulted in a significant reduction in the number of self-FAP expressing cells in the medium. Neither virus resulted in a decrease in FAP ⁺ cells in ascites.

Example 17-Materials and Methods Cell lines HEK293A, DLD, SKOV3, MCF7, A431, A549, and PC3 cells (ATCC) were Dulbecco's ed. From Roswell Park Memorial Institute (RPMI-1640, Sigma-Aldrich, UK). Cultured on 's Medium (DMEM, Sigma-Aldrich, UK) and CHO cells (ATCC). Growth medium is supplemented with 10% (v / v) fetal bovine serum (FBS, Gibco, UK) and 1% (v / v) penicillin / streptomycin (10 mg / mL, Sigma-Aldrich) to humidify the cells. Maintained at 37 ° C., 5% CO 2 in atmosphere. Cells were maintained in DMEM supplemented with 2% FBS for viral infection and viral plasmid transfection. Cells were maintained in DMEM without FBS for recombinant BiTE plasmid transfection. EpCAM expression in the target cell line was determined by flow cytometry.

Generation of EpCAM-expressing stable cell line The protein sequence of the EpCAM gene (SEQ ID NO: 4072) was obtained from DNA synthesized by the NCBI database and Oxford Genetics Ltd (Oxford, UK). The EpCAM gene was cloned into the pSF-Lenti vector by standard cloning techniques that generate a pSF-Lenti-EpCAM vector. HEK293T cells were transfected with Lipofectamine 2000 with lentiviral EpCAM expression vector along with pSF-CMVHIV-Gag-Pol, pSF-CMV-VSV-G, pSF-CMV-HIV-Rev (Oxford Genetics Ltd). The supernatant containing lentivirus was collected after 48 hours and mixed with polybrene (8 μg / mL). The lentivirus / polybrene mixture was added to CHO cells and incubated at 37 ° C. On day 4, the supernatant was replaced with media containing 7.5 μg / mL puromycin. Stable mutants were then clonal selected and EpCAM expression of the parent cell line or stably transfected mutants was determined by antibody staining with EpCAM or an isotopic control antibody and analyzed by flow cytometry. Positive clones were expanded and used for further experiments.

Peripheral blood mononuclear cell (PBMC) preparation and T cell isolation PBMC were isolated by density gradient centrifugation (Boyum, 1968) from whole blood leukocyte cones obtained from NHS Blood and Transplant UK (Oxford, UK). did. The blood was diluted 1: 2 with PBS, layered on Ficoll (1,079 g / mL, Ficoll-Paque Plus, GE Healthcare) and then centrifuged at 400 g for 30 minutes at 22 ° C. with reduced speed. After centrifugation, PBMCs were collected, washed twice with PBS (300 g, 10 minutes at room temperature) and resuspended in RPMI-1640 medium supplemented with 10% FBS. To extract CD3-positive T cells from PBMC, non-CD3 cells were removed using Pan T Cell Isolation Kit (Miltenyi Biotec, # 130-096-535) according to the manufacturer's protocol. To further isolate CD4 and CD8 positive T cells, CD3 T cells were purified one more time using CD4 + Microbeads (Miltenyi Biotec, # 130-045-101).

Primary ascites and pleural effusion primary human malignant ascites and pleural effusion samples after informed consent from patients with multiple indications of advanced cancer including but not limited to ovarian cancer, pancreatic cancer, breast cancer and lung cancer Was received from Churchill Hospital (Oxford, UK) at Oxford University Hospital. The study was approved by the Research Ethics Committee of the Oxford Center for Histopathology. Upon receipt, the cell and liquid fractions were separated and the liquid was used immediately or aliquots were stored at -20 ° C for future analysis. Cell fractions were treated with erythrocyte lysis buffer (Roche, UK) according to the manufacturer's instructions. Cell number and viability were determined by trypan blue staining. The cell types present in each sample were determined by antibody staining for EpCAM, EGFR, FAP, CD45, CD11b, CD56, CD3, CD4, CD8, PD1 and CTLA4 and analyzed by flow cytometry. Fresh cells and fluids were used for ex vivo T cell activation and target cell lysis experiments. In some cases, adherent cells were passaged in DMEM supplemented with 10% FBS and grown for later use.

BiTE engineering and production BiTE was generated by linking two scFvs of different specificities with a flexible GS linker. Each scFv is generated by linking the VH and VL domains from the parent monoclonal antibody with a linker. Each BiTE carried an immunoglobulin light chain (Ig) N-terminal signal sequence for mammalian secretion and a C-terminal decahistidine affinity tag for detection and purification. BiTE was engineered by standard DNA cloning techniques and inserted into a cytomegalovirus (CMV) promoter-driven constitutive protein expression and secretion protein expression vector (pSFCMV-Amp). pSF-CMV-EpCAMBiTE or pSF-CMV-control BiTE plasmid DNA was transfected into HEK293A cells using polyethyleneimine (PEI, Linear, MW 25000, Polysciences, USA) under the following conditions: 55 μg of plasmid DNA: 110 μg PEI (DNA: PEI ratio is 1: 2 (w / w)) was added to the cells, incubated for 4 hours at 37 ° C., then replaced with fresh serum-free DMEM, and further for 48 hours at 37 ° C., 5% CO 2. Incubated. Cells were transfected in parallel with pSF-CMV-GFP to ensure transfection efficiency. To recover the secreted protein, the supernatant of the transfected cells was collected and centrifuged at 350 g, 4 ° C. for 5 minutes to remove cellular components. The supernatant was transferred to a 10,000 MWCO Amicon Ultra-15 centrifugal filter unit (Millipore). After centrifugation at 4750 g at 4 ° C., the volume of retentate was adjusted using a flow-through to obtain a 50-fold higher concentration. An aliquot of the concentrated protein was stored at -80 ° C.

Generation of BiTE-expressed Enadenochirev (EnAdenotucirev) Plasmids pEnAd2.4-CMV-EpCAMBiTE, pEnAd2.4-SA-EpCAMBiTE, pEnAd2.4-CMV-Control BiTE, pEnAd2.4-SA-Control BiTE Transgene cassettes encoding BiTE or control BiTE were generated by direct insertion into the basic EnAd plasmid pEnAd2.4 using Gibson assembly technology. The transgene cassette contains a 5 'short splice acceptor sequence or an exogenous CMV promoter, followed by EpCAM or control BiTE cDNA sequence and 3' polyadenylation sequence downstream. A schematic diagram of the inserted transgene cassette is shown in FIG. The correct construction of the plasmid was confirmed by DNA sequencing. Plasmids EnAd2.4-CMV-EpCAMBiTE, pEnAd2.4-SA-EpCAMBiTE, pEnAd2.4-CMV-control BiTE and pEnAd2.4-SA-control BiTE were restricted by the enzyme AscI before transfection into HEK293A cells. Linearized by digestion. Virus production was monitored by observing cytopathic effects (CPE) in the cell monolayer. Once extensive CPE was observed, virus was recovered from HEK293A cells in 3 freeze-thaw cycles. Single virus clones were selected by serial dilution of the collected lysates, reinfection with HEK293A cells and collection of wells containing a single plaque. To amplify the virus stock, HEK293A cells were serially infected when the infection reached full CPE. Once a strong virus stock was amplified, the virus was purified by double cesium chloride banding to generate EnAd-CMVEpCAMBiTE, EnAd-SA-EpCAMBiTE, EnAd-CMV-control BiTE, EnAd-SA-control BiTE virus stocks. These stocks were titrated by TCID 50 and Pico Green Assay (Life Technologies) according to the manufacturer's instructions.

Supernatant Preparation To assess cytokine release via BiTE, DLD cells (20,000) were treated with 100,000 alone in 96-well flat bottom plates or with 2 ng / μL EpCAM or control BiTE. Plated with CD3 + T cells. After incubation for 48 hours at 37 ° C., 5% CO 2, the supernatant was collected, cellular components were removed by centrifugation, and aliquots were stored at −20 ° C. To evaluate BiTE transgene expression from recombinant viruses, HEK293A (1e6) or DLD cells (1.2e6) were transformed into EnAd-CMV-EpCAMBiTE, EnAd-SA-EpCAMBiTE, EnAd-CMV control BiTE, EnAd-SA. Infected with control BiTE or 100 vp / cell EnAd. Cells were cultured for 72 hours, at which point cytopathic effect (CPE) proceeded. The supernatant was collected and centrifuged at 300 g for 5 minutes to remove cell debris and stored at −20 ° C. for future analysis.
Immunoblotting dot blot was used to determine the concentration of recombinant BiTE produced from plasmid transfection. Two-fold serial dilutions of each BiTE and protein standard (10 × His tagged (C-terminal) human cathepsin D, Biolegend, # 556704) were prepared. The molar concentration of the protein standard was adjusted to represent a BiTE concentration of 100 μg / mL. Each sample and 2 μL of protein standard were applied directly to the nitrocellulose membrane. Air dry membrane and block and probe with α-6xHis (C-terminal) antibody (1: 5000, clone 3D5, Invitrogen, UK, # 46-0693) to detect C-terminal His-tagged protein followed by washing Incubated with anti-mouse secondary antibody (1: 10000, Dako, # P0161) and detected by applying SuperSignal West Dura Extended Duration Substrate (Thermo Fisher, # 34075) according to manufacturer's instructions. The supernatant of virus-infected HEK293A cells was analyzed by Western blotting for BiTE expression. The supernatant was fractionated by SDS-PAGE and transferred to a nitrocellulose membrane according to the manufacturer's protocol (Bio-Rad). The membrane was further processed as in the dot blot protocol described above.

Enzyme-linked immunosorbent assay (ELISA)
To assess EpCAM binding, ELISA plates were prepared by coating overnight at 4 ° C. with human EpCAM / TROP-1 protein (50 ng / well, Sino Biological Inc, # 10694-H02H-50). Plates were blocked with 5% BSA for 1 hour at ambient temperature and then incubated with diluted EpCAM BiTE-, control BiTE- and empty pSF-CMV vector transfected HEK293A supernatant (2 hours, room temperature). The plate was washed 3 times with PBS-T and after each subsequent binding step. Plates with anti-His (C-terminal) antibody (1: 5000, clone 3D5, # 46-0693, Invitrogen, UK) for 1 hour at room temperature, followed by HRP-conjugated anti-mouse Fc (1: 1000, PBS / 5% milk, Dako) and incubated at room temperature for 1 hour. HRP detection was performed using 3.3.5.5′-tetramethylethylenediamine (TMB, Thermo-Fisher) and a stop solution was used to stop the reaction. Absorbance at 450 nm was measured with a Wallac 1420 plate reader (Perkin Elmer).

Flow cytometry Flow cytometry analysis was performed on a FACSCalibur flow cytometer (BD Biosciences) and the data was processed with FlowJo v10.0.7r2 software (TreeStar Inc., USA). For classification of the different cell populations, antibodies specific for CD45 (HI30, Biolegend), CD11b (ICRF44, Biolegend), EpCAM (9C4, Biolegend) and FAP (427819, R & D Systems) were used. For analysis of T cell populations, the following antibody clones bound to different fluorophores were used: CD69 (FN50, Biolegend), CD25 (BC96, Biolegend), IFNγ (4S.B3, Biolegend), αCD107a antibody (H4A3 Biolegend), CD3 (HIT3a, Biolegend), CD4 (OKT4, Biolegend), CD8a (HIT8a, Biolegend), PD1 (H4A3, Biolegend). In each case, an appropriate isotype control antibody was used.

Characterization of human T cell activation CD69 and CD25 expression levels The ability of recombinant EpCAM BiTE or EpCAM BiTE virus to induce T cell activation was assessed by surface expression of CD69 and CD25. Human CD3 cells from PBMC or ascites samples (75,000 cells / well in 96 well flat bottom plates) in the presence of medium alone, EpCAM or control BiTE protein (2 ng / μL) or recombinant virus (100 vp / cell) , Cultured alone or with DLD, SKOV, CHO, CHO pCAM or ascites target cells (15,000). In some cases, anti-PD1 (Invivogen, # hpd1ni-mab7) antibody was added at a final concentration of 2.5 μg / mL. As a positive control for T cell activation, CD3 cells were incubated with CD3 / CD28 Dynabeads (Thermo Fisher, # 11131D). Unless otherwise stated, cells were cultured in medium for 24 hours at 37 ° C. and subsequently harvested using enzyme-free cell dissociation buffer (Gibco, # 13151014). Total cells were stained with antibodies for surface expression of CD69, CD25, CD3, CD4 or CD8 and analyzed by flow cytometry. The effect of ascites on T cell activation (CD69, CD25) was compared with plate-immobilized CD3 antibody (7.5 μg / mL, HIT3a, Biolegend, # 300313) in fluid isolated from RPMI-1640 or malignant ascites samples. CD3 purified PBMC (100,000) were probed by polyclonal activation by incubating.

The ability of IFNγ-expressed EpCAM BiTE to induce T cell activity is indicated by the ability of T cells to become DLD cells (200,000 CD3 cells / well, 40,000 DLD cells / well in flat bottom 96-well plates) and 2 ng / Evaluated by IFNγ expression by co-culture with μL of recombinant EpCAM or control BiTE for 6 hours. As a positive control, T cells were stimulated with a soluble PMA / ionomycin cell activation cocktail (Biolegend, # 4233301). Brefeldin A (GolgiPlug, BD Biosciences) was added to the medium 5 hours prior to harvesting, at which time CD3 + T cells were harvested, stained intracellular for IFNγ expression, and analyzed by flow cytometry.

T cell proliferation To study T cell proliferation, 100,000 CFSE-labeled (CellTrace CFSE kits, Invitrogen, # C34554) CD3 + T cells with 2 ng / μL EpCAM or control BiTE in a 96-well plate format, 20 Incubated with 1,000 DLD cells.

  After 5 days of co-culture, cells were stained with CD3, CD4, or CD8, CFSE fluorescence of viable CD3 + T cells was measured by flow cytometry, and total cell number was normalized using precision counting beads (5000 / well, Biolegend, # 424902). Fluorescence data was analyzed and modeled using the proliferation function of FlowJo v7.6.5 software. Data is expressed as the percentage of original cells that entered the growth cycle (% division) or the average number of cell divisions that were received by the cells of the original population (division index).

DLD cells (15,000 cells / well) were co-cultured with 75,000 CD3 + T cells in flat bottom 96-well plates in the presence of CD107a degranulation medium alone or 2 ng / μL of control or EpCAM BiTE. αCD107a or isotype control antibody was added directly to the medium. Monensin (GolgiStop, BD Biosciences) was added after 1 hour incubation at 37 ° C. and 5% CO 2, followed by another 5 hours incubation. Cells were then harvested, stained for CD3, CD4 or CD8 and analyzed by flow cytometry.

Cytokines in the supernatants taken from cultures of cytokine released DLD / PBMC or pleural effusion cells were quantified using the LEGENDplex human T helper cytokine panel (Biolegend, # 740001) and flow cytometry according to the manufacturer's instructions. Cytokines included in the analysis include IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL- 22, IFNγ and TNFα.

In Vitro Target Cell Cytotoxicity Assay The cytotoxicity of target cells mediated by recombinant BiTE or virus was assessed by LDH release or MTS assay. Target cells (DLD, SKOV, HT-29, A431, A549, PC3, CHO, CHO-EpCAM) in the presence of medium alone, diluted supernatant or virus (100 vp / cell) in a flat bottom 96 well plate, Co-cultured with CD4 or CD8 T cells (E: T 5: 1). After 24 hours of co-culture (unless otherwise indicated), supernatants and cells are collected and cytotoxicity determined by LDH assay (CytoTox 96 non-radioactive cytotoxicity assay, Promega, # G1780) or MTS survival assay (CellTiter 96 Cell Proliferation Assay, Promega, # G3580) according to the manufacturer's instructions. The amount of BiTE produced from virus-infected DLD cells was determined by comparing the cytotoxicity induced by the diluted virus supernatant to that of a standard curve generated using recombinant BiTE.

  To assess the oncolytic activity of the virus, before infecting with increasing vp / cell (5-fold serial dilution, up to 100-5.12e-5vp / cell) or leaving it uninfected, DLD cells were seeded in 96-well plates (25,000 cells / well) at 37 ° C. and 5% CO 2 for 18 hours. On day 5, DLD cytotoxicity was measured by MTS viability assay. A dose response curve was fitted and IC50 determined using a four parameter non-linear fitting model integrated in Prism 7 software (GraphPad Software). Cell viability was monitored in real time using xCELLligence RTCA DP technology (Acea Biosciences). DLD, SKOV3 or MCF7 cells were plated at 12,000 cells / well in 48 well E plates. Plates were incubated at 37 ° C., 5% CO 2 for 18 hours before cells were treated with BiTE (2 ng / μL) or infected with virus (100 vp / cell) or left untreated. Two hours after infection, 75,000 CD3 + cells were added to the required wells. Cell impedance was measured every 15 minutes for up to 160 hours. For ex vivo cytotoxicity assays, unpurified cells from ascites or pleural effusion samples were resuspended in ascites and plated in flat bottom 96 well plates (1.5e5 / well). After incubating at 37 ° C., 5% CO 2 for the stated period, supernatants are analyzed by LDH assay or whole cells are collected with cell dissociation buffer and stained with CD3, CD25, and EpCAM and analyzed by flow cytometry. analyzed. Anti-PD1 antibody (2.5 μg / mL, Invivogen, # hpd1ni-mab7) antibody was included in PD1 blocking experiments.

Viral genome replication and qPCR
The ability of EnAd-CMV-EpCAMBiTE, EnAd-SA-EpCAMBiTE, EnAd-CMV-control BiTE, EnAd-SA control BiTE or EnAd to replicate their respective genomes is 24 wells prior to infection with 100 vp / cell. Plates (150,000 cells / well) were analyzed by seeding at 37 ° C., 5% CO 2 for 18 hours. Wells were harvested 24 and 72 hours after infection and DNA was purified using the PureLink Genomic DNA Mini Kit (Invitrogen, # K182001) according to the manufacturer's protocol. The entire viral genome was quantified by qPCR on EnAd hexon using a specific primer-probe set (primer: TACATGCACCATCGCCGA / CGGGCGGAACTGCACCCA, probe: CCGACTCAGGTACCTCCGAAGCATCCT).

Microscopic examination Bright field and fluorescence images were captured with a Zeiss Axiovert 25 microscope. Time-lapse videos were obtained and observed for viral or T cell-mediated lysis of target cells with EnAd or EnAd-CMVEpCAMBiTE. Uninfected cells were used as a negative control. NHDF cells were stained with CellTracker Orange CMTMR dye (Life Technologies, # C2927), and CD3 + cells were stained with CellTrace Violet Cell Proliferation Kit (Life Technologies, # C34557). Stained NHDF was plated on a 24-well plate at 7,500 cells / well and co-cultured with SKOV3 at 13,500 cells / well. Plates were incubated for 18 hours at 37 ° C., 5% CO 2. Cells were then treated with 300 ng / mL EpCAM BiTE, infected with 100 vp / cell EnAd or EnAd2.4-CMV-EpCAMBiTE, or left untreated. After 2 hours of incubation, 100,000 stained CD3 + was added to the required wells in addition to 1.5 μM CellEvent Caspase 3-7 reagent (Life Technologies, # C10423). Images were taken for 96 hours at 15 minute intervals with a Nikon TE 2000-E Eclipse inverted microscope (10x optical objective). Time-lapse video (12 frames / second) was generated using ImageJ software.

In all cases of two or more experimental conditions being statistically compared, statistical analysis was performed using a one-way analysis of variance test using Tukey's Post Hoc analysis. All data are presented as mean ± SD. The significance levels used were P = 0.01-0.05 (*), 0.001-0.01 (**), 0.0001-0.001 (***). Unless otherwise noted, all in vitro experiments were performed in triplicate.

Example 18-Generation and production of BiTE targeting EpCAM BiTE targeting EpCAM was engineered by linking two scFvs specific for CD3ε and EpCAM with a flexible glycine-serine (GS) linker. . Control BiTE was also produced that recognizes CD3ε and an irrelevant antigen (pertussis filamentous hemagglutinin adhesin (FHA)). Both BiTEs were engineered to include an N-terminal signal sequence for mammalian secretion and a C-terminal decahistidine affinity tag for detection and purification (FIG. 45A). To characterize the function of recombinant BiTE, they were cloned into expression vectors under the transcriptional control of CMV immediate early promoters (pSF-CMV-EpCAMBiTE and pSF-CMV-control BiTE, respectively).

  Adherent HEK293 cells (HEK293A) were transfected with the expression vector and the supernatant was collected and concentrated 50-fold for further analysis. To estimate the amount of BiTE produced, samples were serially diluted and evaluated by dot blot using anti-His and decahistidine-tagged cathepsin D as a standard. In this way, the level of BiTE produced in the supernatant could be estimated to be about 20 μg / mL 48 hours after transfection (1.8e7 HEK293A cells).

  (FIG. 46A). Specific binding of EpCAM BiTE to recombinant EpCAM protein but not control BiTE was shown by ELISA (FIG. 46B).

Example 19-Characterization of human T cell activation by recombinant EpCAM BiTE The ability of recombinant EpCAM BiTE protein to activate PBMC-derived T cells is known to express EpCAM on the surface. Evaluated by adding unstimulated primary human CD3 + cells to cultures of colorectal cancer cells (Karlsson et al., 2008). Addition of 2.5 ng / ml EpCAM BiTE (as supernatant from transduced HEK293A cells) resulted in a significant increase in T cell activation markers CD69 and CD25 (FIGS. 45B and C), whereas control BiTE was effective. There was no.

  Exposure of CD3 cells to EpCAM BiTE in the absence of tumor cells gives a very modest increase in CD69 and CD25, which means that antibody-mediated clustering of CD3 is essential for full activation by this anti-CD3 binding Indicates that T cells stimulated by EpCAM BiTE in the presence of tumor cells also showed a significant increase in gamma interferon production (FIG. 45D) and cell proliferation (FIG. 45E), whereas control BiTE had no effect. The purpose of T cell activation is to cause degranulation-mediated cytotoxicity, and expression of surface CD107a / LAMP1 (Aktas et al., Indicating degranulation) was strongly upregulated by EpCAM BiTE, but depending on the control It was not up-regulated (FIG. 45F).

  Cytokine release after EpCAM BiTE-mediated activation of PBMC-derived T cells in the presence of DLD cells was characterized by flow cytometry using cytokine bead arrays. As before, control BiTE showed little activity, while EpCAM BiTE released several cytokines including high levels of IL-2, IL-6, IL-10, IL-13, gamma interferon and TNF (FIG. 45G). Production of IL-2, gamma interferon, and TNF is generally associated with a Th1 response, but IL-6 and IL-10 are more often associated with a Th2 response (Mosmann & Sad, 1996).

Example 20-Specificity of recombinant EpCAM BiTE Since most human epithelial cells express EpCAM, the effect of EpCAM BiTE

  To assess whether it is antigen-specific, Chinese hamster ovary cells (CHO cells) were engineered with a lentiviral vector to express human EpCAM on the surface. In the presence of EpCAM BiTE and CHO-EpCAM cells, exogenously added PBMC-derived T cells are not strongly activated (evaluated by CD25 expression, see FIG. 47A) and found in parental CHO subject cells and control BiTE Showed related cytotoxicity (FIG. 47B). This indicates that the cytotoxicity of EpCAM BiTE is antigen specific.

  Next, it was evaluated whether EpCAM BiTE kills various tumor cells and whether the level of cytotoxicity mediated by EpCAM BiTE depends on the density of EpCAM expression. T cell cytotoxicity in the presence of EpCAM BiTE was measured in 6 different cancer cell lines, with maximum cytotoxicity observed in DLD and A431, and minimal cytotoxicity observed in A549 and PC3 (FIG. 47C). This showed that A549 and PC3 cells showed the lowest level, DLD showed the highest level, and showed a mild association with the surface level of EpCAM (determined by flow cytometry) (FIG. 47D). This suggests that the presence and level of EpCAM expression affects the degree of cytotoxicity, but other factors (perhaps the cell's intrinsic resistance to granzyme-mediated apoptosis) also affect the overall cell killing level. Play a role in the decision.

Example 21-BiTE-mediated activation of CD4 + and CD8 + T cell subsets To determine which T cell types are activated by EpCAM BiTE, PBMC-derived T cells were incubated with DLD cells and BiTE prior to flow analysis. Was activated using. The percentage of activated CD4 cells was generally slightly higher, but both CD4 + and CD8 + cells showed high levels of expression of CD69 and CD25 (FIG. 49A). EpCAM BiTE-mediated T cell proliferation was assessed using CFSE staining (FIG. 49B), degranulation by expression of CD197a / LAMP1 (FIG. 49C), and again similar levels of activation were seen in both CD4 + and CD8 + cells. It was. Finally, the level of tumor cell cytotoxicity achieved was compared using EpCAM BiTE to activate purified CD4 + and CD8 + subsets. All T cell preparations showed similar cytotoxicity (FIG. 49D), indicating that both CD4 + and CD8 + cells could contribute to the observed BiTE-mediated cytotoxicity.

Example 22-Expression of EpCAM BiTE from the oncolytic adenovirus EnAdenotucirev Enadenocirev (EnAd) is an oncolytic adenovirus and mosaic E2B Group B type 11 and type 3 adenovirus chimeras with a region, an almost complete E3 deletion and a smaller E4 deletion mapped to E4orf4 (Kuhn 2008). This virus, currently undergoing several early-stage clinical trials for cancer treatment, combines excellent systemic pharmacokinetics and promising clinical activity with the potential for transgene coding and expression (Calvo 2014). , Boni 2014). EpCAM BiTE was encoded in EnAd immediately downstream of the fiber gene using a shuttle vector inserted into the viral backbone by Gibson assembly (Figure 18). BiTE was placed under the transcriptional control of the CMV immediate early promoter (EnAd-CMV-EpCAM BITE) or downstream of the splice acceptor site (MLP; EnAd-SA-EpCAM BiTE) of the adenovirus major late promoter. In the former configuration, BiTE should be expressed whenever the virus successfully infects cells, whereas expression from the MLP splice acceptor site activates MLP in cells that permit viral replication. Only happens when you are. Control BiTE (recognizing CD3 and FHA) was also introduced to generate two corresponding control viruses.

  Virus was cloned and rescued in HEK293A cells, and each large batch was prepared in a hyperflask and purified twice by cesium chloride banding. Infection of DLD with parental EnAd and recombinant BiTE virus yielded the same amount of viral genome (measured by qPCR) at all time points tested, indicating that the BiTE transgene did not interfere with viral replication kinetics (Figure 51A). Next, viral replication and oncolytic properties in the absence of human T cells were investigated. DLD cells were infected with virus batches with increasing virus particles (vp) / cell and cytotoxicity was measured by MTS assay on day 5. All recombinant viruses, including those with EpCAM and control BiTE regulated by a CMV promoter or splice acceptor, exhibit cytotoxic activity indistinguishable from the parent virus, and genetic modification alters the virus's intrinsic oncolytic activity. It was shown that there was not (FIG. 51B).

  In order to evaluate BiTE expression and secretion, HEK293A cells were infected with BiTE-expressing EnAd virus, and the supernatant was examined by Western blotting using an anti-His antibody for 72 hours. As shown in FIG. 51C, all four viruses (two expressing control BiTE and two expressing EpCAM BiTE) showed similar levels of BiTE secreted into the supernatant.

Example 23-Selective killing of EpCAM-positive cells with virus-produced EpCAM BiTE EnAd-EpCAM BiTE infected HEK293A cell supernatant was added to the culture medium of CHO and CHO-EpCAM cells with or without PBMC-derived T cells In addition, T cell activation and cytotoxicity against CHO / CHO-EpCAM cells were measured 24 hours later. In the case of CHO cells, no increase in CD25 T cell expression was observed (FIG. 51D) and no cytotoxicity was observed with any treatment (FIG. 51E). However, T cells incubated with CHO-EpCAM cells produced a substantial increase in CD25 expression using supernatants from HEK293A cells infected with either EnAd-CMV-EpCAM BiTE or EnAd-SA-EpCAM BiTE virus. As shown (FIG. 51D). As expected, this is only when CHO-EpCAM cells are added in the presence of supernatant from 293A cells infected with either EnAd-CMV-EpCAM BiTE or EnAd-SA-EpCAM BiTE virus. Converted to selective cytotoxicity against (FIG. 51E). Definitively, there was no cytotoxicity when using supernatants from HEK293A in the absence of T cells or when the cells were infected with EnAd expressing control BiTE.

Example 24-Excellent cytotoxicity of EnAd expressing EpCAM BiTE Some cells, especially SKOV3 ovarian cancer cells, are partially resistant and die more slowly, whereas EnAd directly tumors most cancer cells It is killed quickly by lysis (Kuhn 2008). It was therefore inferred that the results of arming End to secrete EpCAM BiTE resulting in cytotoxic activation of T cells could be particularly evident in SKOV3 cells. Therefore, the cells were exposed to virus (100 vp / cell) 24 hours after seeding and cell death was monitored by the xCELLligence system. PBMC-derived T cells were added (or not added) to the SKOV3 cell culture medium after 2 hours. In the absence of T cells, the tumor cells grew for about 72 hours (as evidenced by the increasing Cell Index signal in FIG. 53A), after which cell growth reached a plateau and was stable for at least 160 hours regardless of viral infection. Was. None of the viruses tested, including the parental EnAd, induced observable target cell cytotoxicity during the time measured. However, when co-cultured with PBMC-derived T cells, both CMV and SA EpCAM BiTE armored viruses have a rapid SKOV3 lysis, such as CMV-driven induction lysis within 16 hours and SA within 44 hours after addition of T cells. Is induced (FIG. 53B). Importantly, parental EnAd or non-specific BiTE control virus did not show target cell lysis within this period even when T cells were added. This result was confirmed by LDH assay in which cytotoxicity was measured at 24, 48 and 96 hours after infection and the same co-culture as described above was set up (FIG. 48). These results are further supported by similar findings in DLD cells where EpCAM BiTE expressing the virus induced cytotoxicity at a significantly faster rate than the control BiTE virus (FIGS. 50A + B).

  To confirm that the target cell cytotoxicity is mediated through T cell activation, CD3 cells were harvested at each time point and the activation status was similar to that observed for cytotoxicity. The expression kinetics were determined by CD69 and CD25 expression (FIGS. 53C and D, FIGS. 50C and D). The approximate amount of EpCAM BiTE produced from infected DLD cells is determined by comparing the cytotoxicity induced by infected DLD supernatant (Abs490) to the cytotoxicity induced by known amounts of recombinant BiTE (Ie, creation of a standard curve (Abs490)). DLD co-cultured with CD3 purified PBMC (1: 5) was incubated with recombinant BiTE (FIG. 50E) or infected DLD supernatant (FIG. 50F) and LDH release was measured at 24 hours. This makes it possible to determine that EpCAM BiTE was produced at 165 μg and 50 μg DLD per million for EnAd-CMV-EpCAMBiTE and EnAd-SAEpCAMBiTE, respectively. EpCAM BiTE has an EC50 of 7.4 ng / ml (FIGS. 50E and F), so EpCAM BiTE is produced by the recombinant virus at levels that are likely to reach therapeutic doses.

  The cytotoxicity of EpCAM BiTE-expressing EnAd was visualized by time-lapse video microscopy. SKOV3 tumor cells (unlabeled) were treated with normal human fibroblasts (EpCAM negative, red labeled) in the presence of a caspase stain (CellEvent Caspase caspase activates the 3-7 reagent to produce a green stain). , Functioning as non-targeting control cells) and PBMC-derived T cells (blue label). In addition, the combination of EpCAM BITE expressing EnAd in combination with exogenous T cells confer dramatic cytotoxicity on SKOV3 tumor cells, which showed strong induction of apoptosis when infected with EnAd-CMV-EpCAMBiTE, but Not shown for EnAd. Importantly, EpCAM-negative NHDF in co-culture was viable throughout. Representative fluorescence images at different time points from the SKOV3 video are shown in FIG. 53E. An equivalent time-lapse video showing DLD cells co-cultured with NHDF, which is inherently more sensitive to viruses, is also shown.

Example 25-EpCAM BiTE overcomes immunosuppression, activates endogenous T cells, and kills endogenous tumor cells in malignant peritoneal ascites and primary fibroblasts (as controls) Clinical samples of 3 malignant peritoneal ascites samples containing non-EpCAM expressing cells) were grown ex vivo and the mixed primary cell populations were incubated with PBMC-derived T cells and free BiTE in culture or 100 vp / cell EnAd -Treated with EpCAMBiTE. After 72 hours, the levels of EpCAM positive target cells (FIG. 55A) or non-target fibroblast activation protein (FAP) positive fibroblasts (FIG. 55B) were measured by flow cytometry. T cell activation was analyzed by measuring CD25 expression (FIG. 55C). Free EpCAM BiTE and EpCAM BiTE-expressing virus induced T cell activation and caused depletion of EpCAM positive tumor cells, while primary FAP positive (EpCAM negative) fibroblasts showed no change in number. This was observed in all patient samples and none of the other treatments had a significant effect. This indicates that EpCAM BiTE (or the oncolytic virus that encodes it) can mediate activation and selective cytotoxicity against human ovarian ascites tumor cells by PBMC-derived T cells.

  Malignant exudates are likely to represent a potential immune tolerance environment in which the immune response commonly seen in late-stage metastatic cancer patients is suppressed. To test this hypothesis, we polyclonally stimulated PBMC-derived T cells in the presence of 100% ascites from five patients with anti-CD3 antibodies or peritoneal malignancies in culture. The anti-CD3 antibody gave about 50% T cell positivity for both CD25 and CD69 in RPMI medium, but the presence of ascites, which appears to attenuate T cell activation as determined by antibody reduction, Mediated an antibody rise in CD25 expression, which was particularly pronounced for patient fluid # 2 (FIG. 56A). This supports our idea that components of ascites can exert immunosuppressive or tolerance effects. However, the attenuation in this increase in activation marker did not correlate with suppression of T cell degranulation, and the expression of CD107a in ascites was similar to that in culture (FIG. 56B). As a result, BiTE can circumvent the tumor microenvironment-related mechanism of T cell immunosuppression (Nakamura & Smith, 2016).

  Thus, the ability of PBMC-derived T cells and EpCAM BiTE to mediate target cell cytotoxicity in the presence of immunosuppressive ascites was investigated. Cytotoxicity showed a delay of about 8 hours in the presence of the patient's ascites # 2, but T cells incubated with ascites 1 and 2 were human mammary carcinoma MCF7 cells as in RPMI medium. Strain lysis was induced (measured using xCELLligence) (FIG. 56C). In addition to the existing immunosuppressive fluid and tumor cells, ascites contains tumor-associated lymphocytes and tumor stroma supporting cells, a unique tumor-like model system for testing BiTE-mediated activation of endogenous patient-derived T cells I will provide a. Total endogenous cells and ascites were incubated with free recombinant BiTE for 24 hours before assessing patient T cell activation (FIG. 56D). In this clinically relevant setting, EpCAM BiTE (which is not a control control) has a lower level of CD25 when tested in 100% ascites than in simple medium, but CD69 and CD25 expression was induced. These data suggest that EpCAM BiTE can overcome at least some immunosuppressive effects of peritoneal ascites to activate endogenous T cells. Cytotoxicity was assessed by measuring the release of LDH, and BiTE caused a significant increase both when the experiment was performed in medium and 100% ascites. This indicates that some ascites cells were killed by BiTE-mediated cytotoxicity, but in the presence of multiple cell types in the primary ascites, defines which part of the tumor cells died It is impossible.

Example 26-EnAd expressing EpCAM BiTE is another clinically relevant situation that can activate endogenous T cells and kill endogenous tumor cells in malignant pleural effusions. In order to investigate the effects of pleural effusion, several samples of pleural effusion were obtained from various malignant tumor patients. At initial screening (example shown in FIG. 52), samples considered suitable for further analysis included CD3 and EpCAM positive cells. PD1 expression by endogenous T cells was also evaluated after initial isolation, indicating that only 10% of PBMC-derived T cells express PD1, whereas all malignant exudate sample T cells are at least 40 relative to PD1. % Positive, sometimes as high as 100% (FIG. 54). Unpurified whole cells (isolated by centrifugation and resuspended) were incubated at a constant concentration in 100% pleural effusion in the presence of 500 ng / mL free EpCAM BiTE or BiTE encoding 100 vp / cell virus. . After 5 days, the total cell population was collected and the total number of CD3 + cells (FIG. 57A) was measured.

  Only samples that received free EpCAM BiTE or EnAd encoding EpCAM BiTE showed T cell proliferation compared to untreated controls. This confirms that EpCAM BiTE bound to the EpCAM target and cross-linked CD3 to stimulate endogenous T cells. The expression level of CD25 on CD3 cells was also determined (FIG. 57B). Free EpCAM BiTE induced significant T cell activation (assessed by CD25 expression) of tumor-associated lymphocytes in all patient samples, even in an environment with high potential for pleural effusion tolerance. In this setting, addition of anti-PD1 blocking antibody had no effect on EpCAM BiTE-mediated T cell activation (FIGS. 54B and C). There was significant variation between patients (although slight between samples from the same patient), but activation ranged from 50% to 90% depending on the donor. Similarly, samples treated with EnAd expressing EpCAM BiTE showed high activation in some patients (from 10-20% for both EnAd-CMV-EpCAM BITE and EnAd-SA-EpCAM BiTE). Up to 80% range).

  Interestingly, patients who showed the lowest BiTE-mediated activation also showed the lowest level of background T cell activation. Parent EnAd, or EnAd expressing control BiTE, or free control BiTE did not cause stimulation above background.

  At the end of the 5 day incubation, the ability of BiTE-expressing virus to mediate EpCAM target cytotoxicity was assessed by measuring residual levels of EpCAM positive cells by flow cytometry (FIG. 57C). Free EpCAM BiTE and the two viruses encoding EpCAM BiTE caused significant depletion of self-EpCAM expressing cells in all cases, but other treatments had little or no effect on the level of EpCAM positive cells. In the case of sample # 1, the survival rate of all EnAd-based viruses is slightly reduced compared to untreated controls, which appears to represent the effect of oncolysis by direct virus. Associated with the lack of influence of PD1 blocking antibody on T cell activation, it has no effect on EpCAM BiTE-mediated killing of target cells and EpCAM + cells in the absence of PD1 blocker (patients 2, 3 and 4) The cytotoxicity of was almost complete. (FIG. 54D).

  The different effects of the parent EnAd and EnAd-CMV-EpCAM BiTE are shown microscopically in FIG. 57D, where BiTE expression reduces the presence of tumor cells and expands the T cell population. The associated flow cytometry plot confirms substantial expansion and activation of T cells after treatment with EpCAM BiTE expressing virus.

  Finally, the effects of various treatments were characterized by measuring the levels of important cytokines produced using the LEGENDplex protein array (FIG. 57E). So far, the largest doubling was gamma interferon, which increased approximately 1000-fold after treatment with free EpCAM BiTE or EnAd encoding EpCAM BiTE. These two treatments also increased the expression of IL-5, IL-13, tumor necrosis factor (TNF), IL17A and IL17F, characteristic of activated T cells, by approximately 10-fold. EnAd alone (or expression of control BiTE) also caused a 10-fold increase in gamma interferon, but other treatments did not cause any significant changes in cytokine expression.

Example 27-Discussion Oncolytic viruses provide an interesting new strategy for combining several therapies within a single targeted self-amplifier (Keller & Bell, 2016; Seymour & Fisher, 2016). Because they grow selectively within cancer cells and spread from cell to cell, some oncolytic viruses are non-apoptotic as part of the process that allows virus particles to escape from dying cells It is believed to mediate cell death through the death pathway (Ingemarsdotter et al., 2010; Li et al., 2013). In particular, EnAd kills cells by a pro-inflammatory process known as necrosis or ischemic cell death (Dyer, 2017). This mechanism of non-apoptotic death is known as ATP, HMGB1, calreticulin (known as damage-related molecular pattern,

  DAMPs) (Weerasinghe & Buja, 2012), which causes the release of several pro-inflammatory cellular components, such as exposure, and is thought to be crucial for the ability of the virus to promote an effective anti-cancer immune response. However, in addition to direct lysis results, the virus encodes and encodes other anticancer biologics while avoiding delivery challenges and ensuring that the biologic achieves its highest concentration within the tumor microenvironment. Provides the possibility to develop. Imlygic encodes GM-CSF, but the possibilities for arming the virus are virtually limitless, and many exciting opportunities to design multimodal therapeutic strategies with additive or synergistic anti-cancer effects (De Gruijl et al., 2015; Hermiston & Kuhn, 2002).

  Encoding BiTE within an oncolytic virus provides a powerful means of activating tumor-infiltrating lymphocytes into cytotoxicity and lysing antigen-positive target cells, completely separate from the effects of direct viral lysis. Provide treatment. In this study we

  Cytotoxicity targeting BiTE is completely antigen-specific, can be mediated by both CD4 and CD8 T cells (Brischwein et al., 2006), incorporated into oncolytic adenoviruses, and viral replication It was shown that it can only be expressed in cells that allow In addition, this study shows that endogenous T cells in liquid cancer biopsies are activated by BiTE and virus-encoded BiTE, killing endogenous tumor cells without additional stimulation or reversal of immune suppression. I showed for the first time what I can do. Importantly, this can also occur in primary body fluids containing the peritoneal ascites or pleural effusion microenvironment as a surrogate for the solid tumor immunosuppressive microenvironment.

  Armed oncolytic viruses for expressing BiTE combine two completely different therapeutic mechanisms, the former providing lytic death of tumor cells that tolerate viral infection, and the latter via T-selected specific antigens. Target cell cytotoxicity. This provides considerable flexibility in the design of therapeutic approaches by using BiTE to deliver cytotoxicity to tumor-associated cells that are likely to be relatively resistant to direct killing by the virus. For example, here we have illustrated a technique using BiTE (EpCAM) that recognizes cancer-associated antigens, but using the BiTE approach to target cytotoxicity against tumor-associated fibroblasts or other stromal cells Is also possible. In fact, even if the target of BiTE recognition is not limited to expression in the tumor microenvironment, linking BiTE production to viral replication can spatially limit BiTE expression to the tumor and reduce systemic toxicity. It can be minimized. This is important because intravenously administered BiTE exhibits relatively short circulatory dynamics (Klinger et al., 2012) and is often associated with significant toxicity on the target but outside the tumor (Teachey et al., 2013). is there.

  The possibility of encoding BiTE within an oncolytic virus has previously been investigated using oncolytic vaccinia virus and Ephrin A2 targeted BiTE. This agent has shown that Ephrin BiTE can mediate PBMC activation and antigen target killing of tumor cells both in vitro and in vivo. Interestingly, BiTE was able to activate T cells, but it did not lead to T cell proliferation unless exogenous IL-2 was added, whereas BiTE used in this study was It produced extensive proliferation of both tumor-associated lymphocytes using in vitro PBMC and ex vivo clinical biopsy samples.

  The observed differences are thought to depend on the different BiTE designs, the different oncolytic viruses used, or perhaps the antigen density that gives sufficient cross-linking of CD3 on T cells.

  One central goal of oncolytic virology is to create an anti-cancer T cell response that recognizes patient specific new antigens and “public” tumor associated antigens. A lytic virus can do this by stimulating improved antigen presentation by lysing tumor cells in the context of DAMP alongside a virus-associated pathogen-associated molecular pattern (PAMP). Immunohistochemical staining of resected colon tumors following intravenous delivery of EnAd suggests that the virus promotes a strong influx of CD8 + T cells into the tumor tissue (Garcia-Carbonero, 2017). However, although this is potentially a very powerful approach, the adaptive T cell response ultimately depends on the expression of MHC class I antigens by tumor cells, allowing targeted killing To do. Loss of MHC expression is a well-proven immune evasion strategy against tumors (Garido et al., 2016).

  It should be noted that both cytotoxic strategies that immediately involve BiTE-armed oncolytic viruses act independently of MHC class I by tumor cells, so that even when tumor cells lose MHC expression, cancer It can be used to kill cells.

  Thus, this study suggests that encoding BiTE within EnAd utilizes a known blood stability and viral systemic bioavailability to achieve a particularly promising strategy to achieve target expression in disseminated tumors. This has been studied in several early phase clinical trials. In particular, in studies in which the virus was administered intravenously several days before resection of the primary colon cancer, subsequent immunohistochemical evaluation of tumor sections revealed that the virus reached the region through the tumor and produced a strong nuclear hexon signal. Showing successful infection and selective viral replication in tumor cells. This corroborates preclinical data (Di et al., 2014; Illingworth, 2017), the virus is stable in 100% human blood, and the tumor target infection of disseminated and metastatic malignancies in human patients. there is a possibility.

  Reflecting the considerable transgene packaging ability of the virus, BiTE can be encoded by EnAd without any loss of oncolytic virulence (FIG. 51B). The presence of the transgene does not affect the physicochemical properties of the virion, so the modified virus should exhibit exactly the same clinical pharmacokinetics as the parent substance, and selectively encode the encoded BiTE within the tumor throughout the body. Should be able to express. This provides an exciting and potentially very effective new approach to systematically targeted cancer immunotherapy that should now be prioritized for clinical evaluation.

Example 28
Immunosuppression of human T cell activation and target cell cytotoxicity by patient malignant exudates malignant exudates are a potential immune tolerance environment in which the immune response commonly observed in late metastatic cancer patients is suppressed Represents. The amount of IL-10, considered to be an anti-inflammatory cytokine, is measured in normal serum or patient's malignant exudate (A, peritoneal ascites, P, pleural effusion) using the human IL-10 ELISA MAX kit (Biolegend, 430604). It was measured. IL-10 levels (88.1-633.4 pg / mL) in the exudate far exceeded the values measured with normal serum (7.2-10 pg / mL). See FIG.

  The ability of CD3 / CD28 beads (Gibco, 11161D) to activate PBMC T cells in the presence of normal serum, ascites or pleural effusion was investigated. Human PBMC T cells (100,000 cells per well in a 96-well plate) were treated with normal serum or CD3 / CD28 beads (according to manufacturer's instructions) in patient exudate (50%). T cells were left untreated in each fluid as a negative control. After 24 hours in culture, cells were harvested and the expression levels of CD69 and CD25 on CD3 + T cells were analyzed by antibody staining and flow cytometry and expressed as the percentage of double positives (CD69 + CD25 + cells) (FIG. 59). In normal serum, anti-CD3 / CD28 beads gave approximately 60% of double positive T cells for both CD25 and CD69, but the presence of ascites caused T cell activation in 6/12 fluid. Weakened.

  In a similar experiment, 100,000 T cells were treated with CD3 / CD28 beads in the presence of normal serum, ascites or pleural effusion (50%). Anti-CD107a or isotype control antibody was added directly to the medium. After 1 hour, monensin was added according to manufacturer's instructions (BD Golgistop, BD Biosciences). After an additional 5 hours, cells were harvested and analyzed by flow cytometry to determine degranulation (FIG. 60). In normal serum, anti-CD3 / CD28 beads gave approximately 22.5% of degranulated T cells, but the presence of ascites attenuated T cell activation in 10/12 fluid. The level of degranulation was significantly correlated with the amount of IL-10 in each fluid (Pearson coefficient, r = −0.7645; p = 0.0038) (FIG. 61).

  In a similar experiment, 75,000 T cells were co-cultured with 15,000 SKOV3 and EpCAM in the presence of normal serum, ascites or pleural effusion (50%). T cells were treated with control BiTE in each fluid as a negative control. After 24 hours of culture, the cells were collected, and the expression levels of CD69 and CD25 on CD3 + T cells were analyzed by antibody staining and flow cytometry and expressed as the percentage of double positives (CD69 + CD25 + cells) (FIG. 62). In normal serum, EpCAM BiTE gave approximately 67.6% of double positive T cells for both CD25 and CD69, but the presence of ascites attenuated T cell activation in 0/12 fluid. Slight activation was induced in 4/10 solution.

  In a similar experiment, 75,000 T cells were co-cultured with 15,000 SKOV3 and EpCAM in the presence of normal serum, ascites or pleural effusion (50%). T cells were treated with control BiTE in each fluid as a negative control. Anti-CD107a or isotype control antibody was added directly to the medium. After 1 hour, monensin was added according to manufacturer's instructions (BD Golgistop, BD Biosciences). After an additional 5 hours, cells were harvested and analyzed by flow cytometry to determine degranulation (Figure 63). In normal serum, EpCAM BiTE beads gave approximately 41.4% of degranulated T cells, but the presence of ascites attenuated T cell activation in 2/12 fluid.

  The ability of EnAd-SA-EpCAMBiTE and EnAd-SA-control BiTE to induce T cell-mediated target cell lysis in malignant exudates was evaluated using xCELLiligence technology. SKOV cells were plated in 48 well E plates at 1e4 cells / well each. Plates were incubated at 37 ° C., 5% CO 2 for 18 hours before cells were infected with 100 virus particles per cell (ppc) or left uninfected. Two hours later, PBMC T cells in normal serum (5: 1) or patient exudates (final, 50%) were added. The cytotoxicity of the target cells was measured every 10 minutes using xCELLligence (FIG. 64). The results suggest that BiTE-mediated SKOV3 lysis by T cells is independent of the fluid used.

  Unpurified ascites cells (and therefore as received) are seeded at 100,000 cells per well of a flat bottom 96 well plate in RPMI medium or ascites. Cells were treated with EpCAM or control BiTE and untreated wells were used as negative controls. After 24 hours incubation at 37 ° C., the cells were harvested and the expression levels of CD25 and CD69 on CD3 cells were determined (FIG. 65). The results demonstrate that EpCAM BiTE results in a significant increase in tumor associated lymphocyte T cell activation (CD69 / CD25 double positive) and is slightly increased by ascites.

  In a similar experiment, unpurified ascites cells (thus unchanged from when received) are seeded at 100,000 cells per well of a flat bottom 96-well plate in RPMI medium or ascites. Cells were treated with EpCAM, control BiTE or recombinant BiTE virus (100 vp / cell) and untreated wells were used as negative controls (FIG. 66). After 5 days incubation at 37 ° C., the total cell population was harvested, the number of CD3 + cells (FIG. 66A) and the expression level of CD25 on CD3 cells (FIG. 66B) were determined, and the flow of endogenous EpCaM + cells by flow cytometry. The number was determined. (FIG. 66C). The total number of cells per well was determined using precision counting beads. The results show that EpCAM BiTE and EnAd expressing EpCAM BiTE resulted in a significant increase in tumor associated lymphocyte T cell activation (CD3 count, CD25) and EpCAM + cell cytotoxicity in both RPMI medium and ascites. Prove that.

  As an extension of the experiment, 6 additional patient exudate samples (7 total) were collected in ascites (FIG. 67), the number of CD3 + determined by flow cytometry (FIG. 67A), CD25 expression of T cells (FIG. 67B). ) And the number of EpCAM + cells (FIG. 67C). The results show that EnAd expressing EpCAM BiTE and EpCAM BiTE significantly increased T cell activation (CD3 count, CD25) of tumor-associated lymphocytes and EpCAM + cell cytotoxicity with good reproducibility. It shows what was brought about in the range.

Example 29
In other experiments in which FAP BiTE mediates T cell activation and FAP + cell death by different donor T cells, the method described in Example 2 was used to test the sets tested in NHDF and T cell co-cultures. The T cell activation properties of the modified FAP BiTE protein were further evaluated compared to control BiTE and polyclonal T cell activation using CD3 / CD28 Dynabeads.

  Supernatants collected after 24 hours of culture were tested with an ELISA for IFNγ (FIG. 68A) and a cytokine bead array (LEGENDplex human T helper cytokine panel, BioLegend # 74001) for a panel of cytokines (FIG. 68B). did. Control BiTE did not induce significant changes in any cytokine, whereas FAP-BiTE resulted in a strong increase in gamma interferon, IL-2, TNFα, IL-17 and IL-10, and the stimulated T cells differed Consistent with the subset, IFNγ production was much greater than that triggered by anti-CD3 / CD28.

Stimulation with FAP BiTE but not control BiTE in the presence of NHDF cells is both CD4 + and CD8 + subsets as determined by CD107a / LAMP1 expression on the T cell surface (assessed by flow cytometry) Also induced rapid degranulation of T cells (within 6 hours), which correlates strongly with their ability to kill target cells (FIGS. 69A and B). This induction of degranulation by FAP BiTE resulted in PBMC T cells (approximately 2.5 ng / mL) with induced T cell activation and cytotoxicity observed with 6/6 donor T cells (FIG. 69D). Converted to strong fibroblast lysis as measured by LDH release after 24 hours of co-culture with EC ₅₀ ) (FIG. 69C). Consistent with the T cells remaining in the inactivated state, cytotoxicity was not induced by control BiTE.

Example 30
Effect of FAP BiTE and EnAd-FAP BiTE virus on cells in primary malignant ascites samples from different ulcer patients As a successor to the study described in Example 16, fresh primary malignant peritoneal ascites from additional cancer patients was replaced with EnAd FAP. Obtained for the study of BiTE virus activity. Three patient samples containing both EpCAM ⁺ tumor cells and FAP ⁺ fibroblasts were expanded ex vivo and the mixed (adherent) cell population was cultured with PBMC-derived T cells and BiTE expressing unmodified or EnAd virus. . After 72 hours, whole cells were harvested and the numbers of FAP ⁺ (FIG. 70A) and EpCAM ⁺ cells (FIG. 70B) were determined by flow cytometry. Furthermore, the activation state of T cells (due to CD25 expression) was measured (FIG. 70C). Infection with both EnAd-CMV-FAPBiTE and EnAd-SA-FAPBiTE induced T cell activation and FAP ⁺ cell depletion in all patient samples, with no significant changes in EpCAM + tumor cell levels. Parental EnAd or control virus did not induce observable T cell activation and the FAP ⁺ cell count remained the same as the uninfected control.

  Importantly, this depletion of FAP + fibroblasts resulted in a consistent and strong decrease in the level of the immunosuppressive cytokine TGFβ detected in the supernatant (FIG. 70D).

In a second series of experiments, total (and unpurified) cells from 5 patient biopsy samples were evaluated to assess the activity of endogenous tumor associated T cells in the samples. Cells were plated in 50% ascites and treated with recombinant control protein or FAP BiTE protein, or 100 vp / cell EnAd or EnAd-BiTE virus. After 5 days of incubation, T cell activation (due to CD25 expression) and the remaining number of FAP ⁺ cells were measured by flow cytometry (FIGS. 71A and B). In all three patient samples, recombinant FAP-BiTE and EnAd-CMV-FAP BiTE induce strong T cell activation, which activates up to about 80% of patient-derived T cells, which are FAP ⁺ fibers Caused significant depletion of blasts. Interestingly, EnAd-SA-FAP-BiTE induced CD25 expression in 2/3 samples with no observable activation or FAP ⁺ cell depletion in patient 1. This is probably because there are insufficient tumor cells for viral infection and production of BiTE protein (EpCAM ⁺ tumor cells were not detected in this sample by flow cytometry), MLP (SA) Consistent with the requirement of tumor cells for driven transgene expression (this is also of T cell activation and FAP + cell depletion by EnAd-SA-FAP-BiTE virus in patient ascites samples shown in FIGS. 42-44. Explain the lack). Taken together, the data indicate that EnAd expressing FAP-BiTE can reproducibly activate tumor-associated T cells to kill endogenous fibroblasts after tumor cell infection.

In another experiment, FAP-BiTE was blocked by blocking PD-1 checkpoints using patient biopsy samples with 73.6% PD-1 positive T cells and 62.9% FAP ⁺ cells PDL1 positive. It was investigated whether activity could be improved (FIG. 72A). Co-culture similar to the above was set to a final concentration of 2.5 μg / mL in the presence or absence of purified blocking mouse IgG2b antibody against human PDL1 (BioLegend, clone 29E.2A3). After 2 days of culture, all cells were collected and measured for residual FAP + cells and T cell activation. By including blocking anti-PDL1 antibody, there was almost complete lysis on day 2 in either setting, without altering FAP + cell depletion (FIG. 72D), CD25 induction (FIG. 72B) and 2-fold higher IFNγ production (FIG. 72C) produced a moderate increase.

Tumor associated lymphocytes (TAL) isolated from ascites of ovarian cancer patients are reported to be rich in PD-1 expression and impaired effector functions including cytotoxicity and IFNg production. Consistent with this, PD-1 expression is 2 more than those in peripheral blood mononuclear cells (PBMC) from 3 healthy donors on CD3 ⁺ cells from 6 cancer patient ascites biopsies. It was twice as high (FIG. 73A). To evaluate the functionality of T cells in these cancer biopsy samples, control NHF cells and unpurified PBMC or ascites cells (% CD3 + cells for each sample are shown in FIG. 73B) or containing FAP BiTE. Co-cultured with Kiyoshi and the supernatant was collected after 5 days and tested for IFNγ by ELISA (FIG. 73C). IFNγ was not induced by control BiTE. Three of the ascites cell samples produced IFNγ at a level similar to that of the PBMC sample, while the other three had diminished responses to FAP BiTE. We next investigate the ability of these T cells to induce BiTE-mediated lysis of NHDF cells. NHDF was plated, PBMC or ascites cells were added along with BiTE-containing supernatant, and cell viability in the culture was monitored in real time using the xCELLigence cytotoxicity assay system. Despite the variability in IFNγ production, all ascites samples induced complete cytotoxicity of NHDF cells when FAP BiTE was added and were generally seen as seen when affected by PBMC. Similar BiTE-mediated NHDF lysis was shown (FIG. 73D).

  To determine whether FAP BiTE can mediate T cell activation in the presence of a patient's malignant exudate sample (all 50%), PBMC T cells were treated with 50% normal human serum in the presence of NHDF cells. Activated with control or FAP BiTE, or with anti-CD3 / CD28 Dynabeads, in either (NS) or different (cell-free) malignant exudate samples. In normal serum, 74% T cells were activated 24 hours after stimulation with anti-CD3 / CD28 beads (double positive for both CD25 and CD69) whereas 3/5 test ascites Significantly reduced T cell activation compared to the response in NS (FIG. 74A). However, when PBMC was cultured with NHDF and stimulated with FAP BiTE, no observable suppression of T cell activation was observed in the presence of any exudate (FIG. 74B), and FAP BiTE overcomes the immunosuppressive mechanism. Have been demonstrated to activate T cells.

Example 31
EnAd-FAPBiTE-mediated oncolysis and T-cell stimulation produce Th1 cytokines including IFNγ, TNFα, and IL-2 by FAP BiTE-mediated activation of T cells that bias CD11b + TAM to a more activated phenotype in patients with ascites , And subsequent removal of FAP ⁺ fibroblasts (and concomitant reduction in TGFβ1) is related to immunosuppression and other shifts in the tumor microenvironment from carcinogenic to anti-tumor activity Finally, the effect on tumor associated macrophages (TAM) in non-isolated ascites cell samples was evaluated. Whole ascites cells of unpurified patients were plated in 50% ascites and treated with free control or FAP BiTE or infected with EnAd-SA control BiTE or EnAd-SA-FAPBiTE virus (100 vp / cell) . In parallel, some cells were treated with IFNγ to induce an activated CD11b myeloid cell phenotype. After incubating for 3 days, the activation state of T cells was first measured. CD25 + cells were measured by flow cytometry and IFNγ secretion was measured by ELISA.

Treatment with FAP BiTE and EnAd-SA-FAPBiTE resulted in approximately 60% of CD3 ⁺ T cells becoming CD25 + (FIG. 75A) and large amounts of IFNγ in the culture supernatant (FIG. 75B). No increase over background was observed for CD25 expression or IFNγ by control BiTE or control virus. To assess TAM polarization, the expression levels of CD64 and CD86 (M1 or “activated” macrophage marker) and CD206 and CD163 (M2 or TAM marker) were measured on CD11b + cells by flow cytometry (FIG. 75C). Treatment with free FAP BiTE or EnAd expressing FAP BiTE induced a more activated phenotype, manifested by a significant increase in CD64 expression, and a strong decrease in CD206 and CD163, and IFNγ was introduced into the culture. Similar to that observed when added.

  In this experiment, treatment with free FAP BiTE or control virus did not induce a clear change in CD86 above background, whereas EnAd expressing FAP BiTE induced a significant increase in CD86 expression, EnAd virus infection and FAP BiTE activity synergize, indicating that primary bone marrow cells can be activated within an inhibitory tumor microenvironment such as the malignant ascites sample tested here. In this study, IFNγ treatment induced a modest decrease in CD86, indicating that the strong increase in CD86 observed with EnAd-SA-FAPBiTE may be due to an IFNγ-independent mechanism.

Example 32
EnAd-FAPBiTE activates tumor-infiltrating lymphocytes and induces ex vivo cytotoxicity in solid prostate tumor biopsies, one of the most realistic preclinical models of diverse tissues, organs and tumors I will provide a. To assess the activity of FAP BiTE expressing virus in this clinically relevant setting, resected human prostate-derived malignant and benign prostate tissue was studied in several pairs by punch biopsy. It was. Initial screening reproducibly showed that prostate tissue had a ring of EpCAM + tumor cells (FIG. 76A) interspersed between large areas of the stroma containing scattered CD8 T cells (FIG. 76B). . FAP staining was seen in fibroblasts adjacent to the tumor area (FIG. 76C).

  Cores were sliced with a vibratome to a thickness of 300 μm and slice cultures were established in the presence of virus (1.5e9 vp / slice) or left uninfected. After 7 days, slices were fixed, paraffin embedded, sectioned, and T cell activation status was assessed by immunohistochemistry (IHC) by staining for CD25 expression (FIG. 76D). Only samples that received EnAd-CMV-FAPBiTE or EnAd-SA-FAPBiTE showed activation of tumor infiltrating T cells as revealed by strong CD25 staining. Neither untreated nor control virus treatment had detectable CD25 positive cells. Supernatants from these slice cultures harvested 4 and 7 days after infection were tested for IFNγ and IL-2 by ELISA, FAP BiTE virus (FIG. 76E) and EnAd-SA-FAPBiTE virus (FIG. 76F). Increased in IFNγ detected from non-benign malignant prostate slice cultures infected with either of the IL-2 detected in cultures containing. EnAd-SA-FAPBiTE induced large amounts of IFNγ that were detectable earlier than CMV-driven FAPBiTE virus.

Example 33-EnAd viruses expressing EpCAM or FAP BiTE Five viruses encoding single BiTE (NG-611, NG-612, NG-613, NG-614, NG-617) were generated (Table 8).
Table 8

¹ SEQ ID NO 55; ² SEQ ID NO 83; ³ SEQ ID NO 84; ⁴ SEQ ID NO 65; ⁵ SEQ ID NO 85; ⁶ SEQ ID NO 86; ⁷ SEQ ID NO 87;

  In each transgene cassette, either the short splice acceptor sequence (SSA, SEQ ID NO: 55) or the longer splice acceptor sequence (SA, SEQ ID NO: 86) was adjacent to the 5 ′ end of the cDNA encoding BiTE. . At the 3 'end of BiTE, the SV40 late poly (A) sequence (PA, SEQ ID NO: 65) was encoded before either the histidine tag (HIS, SEQ ID NO: 41) or untagged. In viruses NG-611, NG-612, NG-613, and NG-617, the anti-CD3 portion of the BiTE molecule was a single-chain variant of mouse anti-human CD3ε monoclonal antibody OKT3.

Viral production plasmid pEnAd2.4 is used to generate plasmids pNG-611, pNG-612, pNG-613, pNG-614 and pNG-617 by direct insertion of synthetic transgene cassettes (SEQ ID NOs: 88-92, respectively) did. The pNG-611 transgene cassette encodes EpCam targeting BiTE (SEQ ID NO: 93), and the pNG-612, pNG-613 and pNG-617 transgene cassettes include FAP targeting BiTE of SEQ ID NO: 94. The pNG-614 transgene cassette encodes a FAP that targets BiTE of SEQ ID NO: 95. Schematic diagrams of the transgene cassette are shown in FIGS. Plasmid DNA construction was confirmed by restriction analysis and DNA sequencing.

  Plasmids pNG-611, pNG-612, pNG-613, pNG-614 and pNG-617 were linearized by restriction digestion with the enzyme AscI to produce the viral genome. The virus was amplified and purified according to the following method.

The digested DNA was purified by phenol / chloroform extraction and precipitated in 300 μl> 95% molecular biology grade ethanol and 10 μl 3M sodium acetate at −20 ° C. for 16 hours. The precipitated DNA was pelleted by centrifuging at 14000 rpm for 5 minutes, washed with 500 μl of 70% ethanol, and then centrifuged again at 14000 rpm for 5 minutes. Clean DNA pellets were air dried and resuspended in 500 μl OptiMEM containing 15 μl Lipofectamine transfection reagent and incubated at room temperature for 30 minutes. The transfection mixture was then added dropwise to a T-25 flask containing 293 cells grown to a confluence of 70%. After incubating the cells with the transfection mixture for 2 hours at 37 ° C., 4 ml of 5% CO ₂ cell medium (DMEM high glucose with glutamine supplemented with 2% FBS) was added to the cells, and the flask was incubated at 37 ° C., 5% CO 2. Incubated with ₂ .

  Transfected 293 cells were monitored every 24 hours and supplemented with additional media every 48-72 hours. Virus production was monitored by observing significant cytopathic effects (CPE) in the cell monolayer. Once extensive CPE was observed, virus was harvested from 293 cells by three freeze-thaw cycles. To amplify the virus stock, 293 cells were reinfected with the harvested virus. Viable virus production during amplification was confirmed by observation of significant CPE in the cell monolayer. Once CPE was observed, virus was harvested from 293 cells by three freeze-thaw cycles. The amplified virus stock was used for further amplification before the virus was purified by double cesium chloride banding to produce a purified virus stock.

Viral activity assessed by qPCR A549 cells infected or left uninfected for 72 hours with 1ppc of NG-611, NG-612, NG-617, enadenotucilv, by qPCR Used for quantification of viral DNA. Cell supernatants were collected and clarified by centrifugation at 1200 rpm for 5 minutes. DNA was extracted from 45 μL of supernatant using Qiagen DNeasy kit according to manufacturer's protocol. A standard curve using Enadenotucilv virus particles (2.5e10-2.5e5vp) was also generated and extracted using the DNeasy kit. Each extracted sample or standard was analyzed by qPCR using a viral gene specific primer-probe set to the early gene E3.

  Quantification of the number of detected viral genomes per cell demonstrated that NG-611, NG-612, and NG-617 showed significant genomic replication in the A549 cell line (FIG. 77D). This is the same for all viruses tested, including the parental virus enadenochucil, indicating that inclusion of the BiTE transgene does not affect viral replication activity. No viral genome was detected in uninfected cells (data not shown).

Virus expressing BiTE mediated T cell activation and degranulated cancer cell infected A549 cells were seeded in 24-well plates at a density of 2.5e5 cells / well. Plates were incubated for 4 hours at 37 ° C., 5% CO ₂ before cells were infected or left uninfected with 1 ppc NG-611, NG-612, enadenotucirev. . Supernatants were collected from the cells at 24, 48 or 72 hours after infection, clarified by centrifugation at 1200 rpm for 5 minutes, and snap frozen.

T-cell assay Lung fibroblast cell line MRC-5 expressing FAP, or ovarian cancer cells expressing EpCam, SKOV3, seeded in 48-well plates at a density of 5.7e4 cells / well and 1.2e5 cells / well, respectively did. After incubating the plate for 4 hours at 37 ° C., 5% CO ₂ , the medium was replaced with 150 μL / well thawed supernatant recovered from the A549 plate. Purified CD3 T cells isolated from human PBMC donors were then also added to the plates to obtain a 2: 1 T cell to MRC-5 or SKOV3 ratio. After incubating the co-culture for 16 hours at 37 ° C., 5% CO ₂ , cell supernatants were collected for ELISA analysis and T cells were collected for flow cytometry analysis. The medium containing non-adherent cells was removed from the co-culture wells and centrifuged (300 × g). The supernatant was carefully removed, diluted 1/2 with PBS 5% BSA and saved for ELISA analysis. The adherent cell monolayer was washed once with PBS and then detached using trypsin. Trypsin was inactivated using 10% FBS RPMI medium and cells were added to the cell pellet collected from the culture supernatant. The cells were centrifuged (300 × g), the supernatant was discarded and the cell pellet was washed in 200 μL PBS. The cells were centrifuged again and then resuspended in 50 μL PBS with Live / Dead Aqua (Life technology) for 15 minutes at room temperature. Cells were washed once in FAC buffer before staining with a panel of direct binding antibodies: anti-CD3 conjugated to AF700; anti-CD25 conjugated to BV421; anti-HLA-DR conjugated to PE / CY5 Anti-CD40L conjugated to BV605; anti-CD69 conjugated to PE and anti-CD107a conjugated to FITC. Samples of cells from each co-culture condition were also stained with the relevant isotype control antibody. All staining was performed in FAC buffer at a total volume of 50 μL / well for 15 minutes at 4 ° C. Cells were then washed twice with FAC buffer (200 μL), resuspended in 200 μL FAC buffer and analyzed by flow cytometry (Attune).

Up-regulation of T cell activation markers Flow cytometric analysis of T cell activation is based on the expression of T cell activation markers CD25, CD69, HLA-DR and CD40L or T cell degranulation marker CD107a on living single cells. evaluated. These data show that when NG-611 supernatant was added to the cells when co-cultured with EpCam + SKOV3 cells, CD25, compared to NG-612, enadenocileev or untreated control supernatants. It was shown that the number of T cells expressing CD69, HLA-DR, CD40L or cell surface CD107a was significantly increased (FIG. 78). For all these markers, the supernatant from A549 cells infected for 24 hours hardly stimulated T cell activation, but by 48 hours post infection the supernatant had significant T cell activity across all markers. Stimulated. This was the same at 72 hours after infection.

When co-cultured with FAP ⁺ MRC-5 cells, the addition of NG-612 supernatant to the cells resulted in CD25, compared to NG-611, enadenotulev or untreated control supernatant. The number of T cells expressing CD69, HLA-DR, CD40L or cell surface CD107a was significantly increased (FIG. 79). Some T cell activation, possibly due to low but detectable expression of EpCam (about 5%) on the MRC-5 cell line that binds EpCam BiTE expressed by NG-611 virus is also observed with NG-611 virus (FIG. 80). For all these markers, the supernatant from A549 cells infected for 24 hours hardly stimulated T cell activation, but by 48 hours post infection the supernatant had significant T cell activity across all markers. Stimulated. After incubation with the supernatant collected 72 hours after infection, CD25 and CD69 markers were also up-regulated, but in the supernatant collected 72 hours after infection, the activation markers, HLA-DR, CD40L and CD107a were 48 Detected at a level lower than time. This is due to the high levels of BiTE present at this late stage of infection, resulting in rapid and potent T cell activation and the need to measure effector function at a time earlier than 16 hours after incubation with the supernatant. It means that there is.

  For detection of IFNγ expression, the co-culture supernatant was diluted in 5% BSA / PBS assay buffer (within a range of 1:10 to 1: 1000) and the ELISA was replaced with a human IFN-gamma Quantikine ELISA kit (R & D systems). And performed according to the manufacturer's protocol. The concentration of secreted IFNγ was determined by interpolation from a standard curve. IFNγ expression can only be detected in the supernatant of co-cultures using NG-611 on SKOV3 cells (FIG. 81A) or NG-611, NG-612 on MRC-5 cells (FIG. 81B). It was.

Example 34: Immunoactivation and anti-tumor effect of BiTE expressing virus in vivo NSG mouse humanized CD34 + hematopoietic stem cells (from Jackson Labs) together with HCT116 tumor cells subcutaneously on both flank 18 weeks after engraftment Transplanted. When tumors reached 80-400 mm ³ , mice were grouped so that each treatment group had an equivalent tumor volume distribution, with 7 mice per group. Mice were injected intra-tumorally with either saline, enadenoticirev or NG-611, 5 × 10 ⁹ particles per injection, 2 injections per tumor. The tumor on both sides was treated. Tumor volume was measured 3 to 4 times a week, and significant anti-tumor responses were compared with enadenoticirev or untreated controls for up to 20 days after NG-611 treatment. Has been demonstrated (FIG. 82a). Twenty days after dosing, one tumor from each group of 4 mice was processed for flow cytometry, while the remaining tumors were frozen on dry ice.

Flow cytometric tumor samples were mechanically separated in a small amount of RPMI medium immediately after excision. The separated tumor was then passed through a 70 μm cell strainer and centrifuged at 300 g for 10 minutes. The cell pellet was resuspended in 100 μL PBS containing Live / Dead Aqua (Life technology) for 15 minutes on ice. Prior to staining with a panel of direct binding antibodies, cells were washed once with FACs buffer (5% BSA PBS): anti-CD8 (RPA-T8, AF700); anti-CD4 (RPA-T4, PE); anti-CD45. (2D1, APC-Fire 750); anti-CD3 (OKT3, PerCP-Cy5.5); anti-CD25 (M-A251, PE-Dazzle 594); anti-CD69 (FN50, APC); anti-HLA-DR (L243, BV605) ); Anti-CD107a (H4A3, FITC). Tumor cell suspension pools were also stained with related isotype control antibodies. All staining was performed in FAC buffer in a total volume of 50 μL / well for 20 minutes at 4 ° C. Cells were washed 3 times with FAC buffer (200 μL), then resuspended in 200 μL FAC buffer and analyzed by flow cytometry (Attune). FAC analysis showed that the ratio of CD8 T cells to CD4 T cells in the tumor was significantly increased in NG-611 treated tumors compared to enadenotucilv treated or untreated controls. (FIG. 82b).

Example 35-EnAP virus FAP BiTE coexpressing FAP BiTE and immunoregulatory cytokines and chemokines and three viruses encoding immunmodulatory proteins (NG-615, NG-640 and NG-641) were generated (Table 9) ).
Table 9

¹ SEQ ID NO 55; ² SEQ ID NO 87; ³ SEQ ID NO 63; ⁴ SEQ ID NO 105; ⁵ SEQ ID NO 61; ⁶ SEQ ID NO 107; ⁷ SEQ ID NO 64; ⁸ SEQ ID NO 109; ⁹ SEQ ID NO 65; ¹⁰ SEQ ID NO 110; ¹¹ SEQ ID NO: 111; ¹² SEQ ID NO: 86

Plasmids pNG-615, pNG-616, pNG-640 and pNG-641 were generated by direct insertion of synthetic transgene cassettes (SEQ ID NOs: 112-114, respectively) using the viral production plasmid pEnAd2.4. NG-615 and NG-616 contain four transgenes encoding FAP target BiTE (SEQ ID NO: 94), Flt3L (SEQ ID NO: 115), MIP1α (SEQ ID NO: 116) and IFNα (SEQ ID NO: 117). NG-640 and NG-641 encode the FAP targets BiTe (SEQ ID NO: 94), CXCL9 (SEQ ID NO: 118) and CXCL10 (SEQ ID NO: 119), and NG-641 also encodes IFNα (SEQ ID NO: 117). Contains a fourth transgene. Schematic diagrams of the transgene cassette are shown in FIGS. Plasmid DNA construction was confirmed by restriction analysis and DNA sequencing.

  Plasmids pNG-615, pNG-616, pNG-640 and pNG-641 were linearized by restriction digestion with the enzyme AscI to generate the viral genome. Virus was amplified and purified according to the method detailed in Example 33.

Virus-activated cancer cell infection assessed by qPCR and transgene ELISA 1ppc of NG-615, adenatucirev infected with A549 cells for 72 hours or left uninfected Used for DNA quantification and analysis of transgene expression by ELISA. Cell supernatants were collected and clarified by centrifugation at 1200 rpm for 5 minutes. 45 μL of supernatant was used for DNA analysis and the remaining supernatant was used for ELISA.

qPCR
DNA was extracted from the supernatant samples using the Qiagen DNeasy kit according to the manufacturer's protocol. A standard curve using Enadenotucilv virus particles (2.5e10-2.5e5vp) was also generated and extracted using the DNeasy kit. Each extracted sample or standard was analyzed by qPCR using a viral gene specific primer-probe set to the early gene E3. Quantification of the number of detected viral genomes per cell demonstrates that NG-615 showed significant genomic replication at the same level as the parental virus adenanotucilev in the A549 cell line (FIG. 84). These data indicated that inclusion of BiTE and the three immunoregulatory transgenes did not significantly affect viral replication activity. No viral genome was detected in uninfected cells.

ELISA
The IFNα ELISA was performed using the Verikine Human IFN alpha kit (Pbl assay science), the MIP1α ELISA was performed using the human CCL3 Quantikine ELISA kit (R & D systems), the Flt3L ELISA kit, and the Flt3L ELISA kit. Abcam). All assays were performed according to the manufacturer's protocol.

  The concentration of secreted IFNα, MIPα or FLt3L was determined by interpolation from a standard curve. Expression of IFNα, MIP1α and Flt3L was detected in the cell supernatant of NG-615 but not in enadenotucilv or untreated control cells (FIG. 85).

Virus expressing BiTE mediated T cell activation and degranulated cancer cell infected A549 cells were seeded in 24-well plates at a density of 2.5e5 cells / well. Plates were incubated for 4 hours at 37 ° C., 5% CO ₂ before cells were infected or left uninfected with 1 ppc NG-612, NG-615, enadenotucirev. . Supernatants were collected from the cells at 24, 48 or 72 hours after infection, clarified by centrifugation at 1200 rpm for 5 minutes, and snap frozen.

T cell assay Lung fibroblast cell line MRC-5 expressing FAP was seeded in 48 well plates at a density of 5.7e4 cells / well. After incubating the plate for 4 hours at 37 ° C., 5% CO ₂ , the medium was replaced with 150 μL / well thawed supernatant recovered from the A549 plate. Purified CD3 T cells isolated from a human PBMC donor were then also added to the plate to give a 2: 1 T cell to MRC-5 ratio. Cell supernatants are collected for ELISA analysis and the co-cultures are incubated for 16 hours at 37 ° C., 5% CO ₂ before harvesting T cells for flow cytometric analysis according to the method detailed in Example 29. Incubated.

Up-regulation of T cell activation markers Flow cytometric analysis of T cell activation was performed on T cell activation markers CD25, CD69, HLA-DR and CD40L on live CD3 + single cells, or T cell degranulation marker CD107a. Evaluated by expression. These data show that when co-cultured with FAP ⁺ MRC-5 cells, the addition of NG-615 or 612 supernatant compared to enadenoticil or untreated control supernatants compared to CD25, It showed that the number of T cells expressing CD69, HLA-DR, CD40L or CD107a was significantly increased (FIG. 86).

For detection of secreted IFNγ expression of the stimulatory cytokine IFNγ, the co-culture supernatant was diluted in 5% BSA / PBS assay buffer (within a range of 1:10 to 1: 1000) and the ELISA was human IFN-gamma Quantikine kit (RandD Systems) and according to the manufacturer's protocol. The concentration of secreted IFNγ was determined by interpolation from a standard curve. The expression of IFNγ could only be detected in the supernatant of co-cultures using A549 supernatant infected with NG-612 or NG-615 (FIG. 87).

Example 36-EnAd virus co-expressing BiTE targeting FAP and BiTE targeting EpCam One targeting EpCam (EpCam BiTE) and the other targeting FAP (FAP BiTE) Two BiTE molecules The encoding virus NG-618 was generated (Table 10).
Table 10

¹ SEQ ID NO: 55; ² SEQ ID NO: 121; ³ SEQ ID NO: 106; ⁴ SEQ ID NO: 122; ⁵ SEQ ID NO: 65;

Plasmid pNG-618 was generated by direct insertion of a synthetic transgene cassette (SEQ ID NO: 123) using the virus production plasmid pEnAd2.4. The NG-618 virus contains two transgenes that encode EpCam target BiTE (SEQ ID NO: 93) and FAP target BiTE (SEQ ID NO: 95). A schematic diagram of the transgene cassette is shown in FIG. Plasmid DNA construction was confirmed by restriction analysis and DNA sequencing.

  Plasmid pNG-618 was linearized by restriction digestion with the enzyme AscI to produce the viral genome. Virus was amplified and purified according to the method detailed in Example 33.

Virus expressing BiTE mediated T cell activation and degranulated cancer cell infected A549 cells were seeded in 6-well plates at a density of 1.2e6 cells / well. Before leaving the cells NG-611, NG-612, NG-618, Ena de Bruno Chu sheet or to infect Rev (enadenotucirev), or uninfected, plates 37 ° C., in 5% CO ₂ 4 hours Incubated. At 72 hours post infection, the supernatant was collected from the cells and clarified by centrifugation at 1200 rpm for 5 minutes.

T cell assay LAP fibroblast cell line MRC-5 expressing FAP and A549 cells expressing EpCam were seeded in 24-well plates at a density of 1.5e5 cells / well. MRC-5 and A549 cells were also mixed at a 1: 1 ratio and seeded in 24 plates at a total cell density of 1.5e5 cells / well. The plate was incubated for 4 hours at 37 ° C., 5% CO ₂ before replacing the medium with 300 μL / well thawed supernatant collected from the A549 plate. Purified CD3 T cells isolated from human PBMC donors were then also added to the plates to give a 2: 1 T cell to MRC-5 or SKOV3 cell ratio. Cell supernatants were collected for ELISA analysis and the co-cultures were incubated for 16 hours at 37 ° C., 5% CO ₂ before collecting T cells, MRC-5 cells and A549 cells for flow cytometry analysis.

Detection of FAP and EpCam in MRC-5 or SKOV cells Flow cytometric analysis of detectable FAP or EpCam on the surface of MRC-5 or SKOV cells, respectively, can be performed before staining cells with a panel of directly bound antibodies. Evaluated by washing once in FAC buffer: anti-FAP conjugated with AF647; anti-EpCam conjugated with PE. The analysis shows that FAP expression is no longer detectable in MRC-5 cells incubated with supernatant from cells infected with virus expressing FAP-BiTE, NG-618, but from EnAd-treated or untreated cells. It was detected on> 80% of the cells incubated with clear. (FIG. 89A). These data indicate that FAP-BiTE produced by the NG-618 virus binds to its FAP target on MRC-5 cells which prevents anti-FAP antibody binding. Live large single cell SKOV cells were evaluated for detectable EpCam expression. In SKOV cells incubated with supernatant (17% cells) infected with virus expressing EpCam-BiTE, NG-618, EpCam expression was only detected at low levels, but cells treated with EnAd Or detected on> 40% of cells incubated with supernatant from untreated cells (FIG. 89B). Taken together, these data indicate that NG-618 produces BITE molecules that bind to EpCam and FAP target proteins.

Up-regulation of T cell activation markers Flow cytometric analysis of T cell activation was performed on T cell activation markers CD25, CD69, HLA-DR and CD40L on live CD3 + single cells, or T cell degranulation marker CD107a. Evaluated by expression. These data indicate that when co-cultured with FAP ⁺ MRC-5 cells, when NG-618 supernatant was added to the cells, CD25 compared to enadenotucilv or untreated counterpart supernatants. The number of T cells expressing CD40L or CD107a was significantly increased (FIG. 90). When NG-618 supernatant is added to EpCam ⁺ SKOV3 cells, the number of T cells expressing CD25, CD40L or CD107a is also compared to the enadenotucilv or untreated control supernatant. There was a significant increase (FIG. 91). These data demonstrate that both BiTE molecules expressed by the NG-618 virus are functional with respect to induction of T cell activation.

Analysis of T cell mediated target (MRC-5 and SKOV) cell killing Flow cytometric analysis of MRC-5 and SKOV cell viability was performed in 50 μL PBS with Live / Dead Aqua (Life technology) for 15 minutes at room temperature. The cells were evaluated by staining. Cells were washed once in FAC buffer before staining with a panel of direct binding antibodies: anti-FAP conjugated to AF647; anti-EpCam conjugated to PE. MRC-5 and SKOV cell viability was significantly reduced after incubation with NG-618 supernatant samples, but significant cell death was detected in enadenotucirev or untreated control supernatants (Figure 92). These data demonstrate the functional ability of NG-618 co-expressed FAP and EpCam target BiTE to induce T cell mediated cell death of target cells.

Sequence number 25: FAP BiTE-P2A-RFP (Italic type = leader, bold = furin cleavage site, underline = P2A sequence, lower case = RFP)
MGWSCIILFLVATATGVHSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSGGGGSDVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGY NYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIKHHHHHHHHHHRRKRGSG ATNFSLLKQAGDVEENPGP mselikenmhmklymegtvnnhhfkctsegegkpyegtqtmkikvveggplpfafdilatsfmygskafinhtqgipdffkqsfpegftwerittyedggvltatqdtsfq ngciiynvkingvnfppsngpvmqkktrgweantemlypadgglrghsqmalklvgggylhcsfkttyrskpkaklnlkmpgfhfvdhrreikeadekethevalkhemdeva
SEQ ID NO: 26: Control (anti-FHA) BiTE-P2A-RFP (italic font = leader, bold = furin cleavage site, underline = P2A sequence, lower case = RFP)
MGWSCIILFLVATATGVHSELDIVMTQAPASLAVSLGQRATISCRASKSVSSSGYNYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREFPLTFGAGTKLEIKSSGGGGSGGGGGGSSRSSLEVQLQQSGPELVKPGASVKISCKTSGYTFTGYTMHWVRQSHGKSLEWIGGINPKNGGIIYNQKFQGKATLTVDKSSSTASMELRSLTSDDSAVYYCARRVYDDYPYYYAMDYWGQGTSVTVSSAKTTPPSVTSGGGGSDVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQG EWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIKHHHHHHHHHHRRKRGSG ATNFSLLKQAGDVEENPGP mselikenmhmklymegtvnnhhfkctsegegkpyegtqtmkikvveggplpfafdilatsfmygskafinhtqgipdffkqsfpegftwerittyed ggvltatqdtsfqngciiynvkingvfpsngpvmqkktrgweantemlypadgglrghsqmalklvgglyhcsfkttyrskkpkaklnlkmpghftlkhekhvkh
SEQ ID NO: 33: Splice acceptor sequence CAGG
SEQ ID NO: 55: short splice acceptor (SSA) DNA sequence (null sequence)
CAGG
SEQ ID NO: 58: Kozak sequence (null sequence)
CCACC

Claims (47)

Formula (I):
5'ITR-B1-BA-B2-BX-BB-BY-B3-3'ITR (I)
Where
B1 is a bond or comprises E1A, E1B or E1A-E1B;
BA includes -E2B-L1-L2-L3-E2A-L4,
B2 is a bond or comprises E3;
BX is a bond or a DNA sequence comprising a restriction site, one or more transgenes, or both;
BB includes L5,
BY is a bond or a DNA sequence containing a restriction site, one or more transgenes, or both;
B3 is a bond or comprises E4;
Here, the adenovirus encodes a bispecific T cell engager (BiTE) comprising at least two binding domains, wherein at least one of said domains is on a subject's immune cells, such as a subject's T cells. Specific for surface antigens of
An adenovirus comprising a sequence of formula (I), wherein the adenovirus is EnAd or Ad11.   The adenovirus according to claim 1, wherein the adenovirus is EnAd.   The adenovirus according to claim 1 or 2, wherein the surface antigen is a component of a T cell receptor complex (TCR) such as one selected from CD3, TCR-α and TCR-β.   4. Adenovirus according to claim 3, wherein the surface antigen is CD3, such as CD3ε, CD3γ and CD3δ, in particular CD3ε.   3. Adenovirus according to claim 1 or 2, wherein one of the binding domains in the BiTE is specific for non-TCR activating proteins such as CD31, CD2 and CD277. One of the binding domains is CEA, MUC-1, EpCAM, HER receptor HER1, HER2, HER3, HER4, PEM, A33, G250, carbohydrate antigens Le ^y , Le ^X , Le ^b , PSMA, TAG-72. The adenovirus according to any one of claims 1 to 5, which is specific for tumor antigens such as STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2 and ErbB3.   The adenovirus of claim 6, wherein one of the binding domains is specific for EpCAM.   One of the binding domains is a tumor stromal antigen, such as fibroblast activation protein (FAP), TREM1, IGFBP7, FSP-1, platelet derived growth factor-α receptor (PDGFR-α), platelet derived growth factor-β The adenovirus according to any one of claims 1 to 7, which is specific for a receptor (PDGFR-β) and vimentin.   9. The adenovirus of claim 8, wherein one of the binding domains is specific for FAP.   The adenovirus according to claim 8 or 9, wherein the stromal antigen is an antigen selected from bone marrow-derived suppressor cell antigen, tumor-associated macrophage, and combinations thereof.   The adenovirus according to claim 10, wherein the antigen is selected from CD163, CD206, CD68, CD11c, CD11b, CD14, CSF1 receptor, CD15, CD33, CD66b and combinations of two or more thereof.   The adenovirus according to any one of claims 1 to 11, wherein at least one of BX and BY is not a bond.   The adenovirus according to any one of claims 1 to 12, wherein the adenovirus is a chimera.   The adenovirus according to any one of claims 1 to 13, wherein the adenovirus is oncolytic.   The adenovirus according to any one of claims 1 to 14, which is capable of replicating the adenovirus.   The adenovirus of claim 15, wherein the adenovirus is capable of replication.   The adenovirus according to any one of claims 1 to 14, wherein the adenovirus is replication-defective.   The adenovirus according to any one of claims 1 to 17, wherein BX comprises one or more transgenes or transgene cassettes.   The adenovirus according to any one of claims 1 to 18, wherein BY comprises one or more transgenes or transgene cassettes.   21. The adenovirus according to any one of claims 1 to 19, wherein the one or more transgenes or transgene cassettes are under the control of an endogenous or exogenous promoter such as an endogenous promoter.   21. The adenovirus according to claim 20, wherein the transgene or transgene cassette is under the control of an endogenous promoter selected from the group consisting of an E4 promoter and a major late promoter, in particular a major late promoter.   The adenovirus according to claim 20 or 21, wherein the transgene or transgene cassette is under the control of an exogenous promoter such as CMV.   23. Adenovirus according to any one of claims 1 to 22, wherein BiTE has a short half-life, for example 48 hours or less.   24. The adenovirus according to any one of claims 1 to 23, wherein BiTE is encoded in a region selected from E1, E3, BX, BY and combinations thereof.   25. The adenovirus according to claim 24, wherein BiTE is encoded at least at position BX, for example under the control of an exogenous promoter.   26. The adenovirus according to claim 24 or 25, wherein BiTE is encoded at least in the BY position, for example under the control of a major late promoter.   27. The adenovirus according to any one of claims 1 to 26, wherein the adenovirus further encodes a second BiTE, for example, both BiTEs are encoded at the BY position.   The first BiTE molecule is specific for a tumor antigen, eg, a tumor antigen, and the second BiTE molecule is on a tumor stromal antigen, eg, a stromal antigen on fibroblasts or a tumor-associated macrophage or bone marrow-derived suppressor cell 28. Adenovirus according to any one of claims 1 to 27, which is specific.   The encoded BiTE comprises a VH domain comprising the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence that is at least 95% identical to it, and an amino acid sequence set forth in SEQ ID NO: 9 or a sequence that is at least 95% identical to it The adenovirus according to any one of claims 1 to 28, comprising VL.   30. The adenovirus of claim 29, wherein the encoded BiTE comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO: 7 or a sequence at least 95% identical thereto.   The encoded BiTE comprises a VH domain comprising an amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence at least 95% identical thereto, and an amino acid sequence set forth in SEQ ID NO: 7 or a sequence at least 95% identical thereto 31. The adenovirus according to any one of claims 1 to 30, comprising VL.   32. The adenovirus of claim 31, wherein the encoded BiTE comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO: 7 or a sequence that is at least 95% identical thereto.   A VH domain wherein the encoded BiTE comprises the amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence that is at least 95% identical thereto, and the amino acid sequence set forth in SEQ ID NO: 12 or a sequence that is at least 95% identical thereto The adenovirus according to any one of claims 1 to 32, comprising VL.   34. The adenovirus of claim 33, wherein the encoded BiTE comprises an scFv comprising an amino acid sequence set forth in SEQ ID NOs: 11, 74, 75, or a sequence that is at least 95% identical to any one thereof.   A VH domain wherein the encoded BiTE comprises the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence that is at least 95% identical thereto, and the amino acid sequence set forth in SEQ ID NO: 17 or a sequence that is at least 95% identical thereto The adenovirus according to any one of claims 1 to 34, comprising VL.   36. The adenovirus of claim 35, wherein the encoded BiTE comprises an scFv comprising an amino acid sequence set forth in SEQ ID NOs: 16, 72, 73, or a sequence that is at least 95% identical to any one thereof.   25. The adenovirus of claim 1, 33 or 24, wherein the adenovirus comprises the sequence set forth in SEQ ID NO: 36, 27, 81, 97, 98, 99 or 100.   37. The adenovirus according to claim 1, 35 or 36, wherein the adenovirus comprises the sequence shown in SEQ ID NOs: 34, 35, 79, 80, 96.   39. Adenovirus according to any one of claims 1 to 38, wherein the adenovirus encodes at least one further transgene, for example 1, 2, 3 or 4 further transgenes.   40. The adenovirus of claim 39, wherein the additional transgene encodes a cytokine, chemokine and / or immunomodulator, eg, encoded at the BY position.   At least one additional transgene is for example MIP1α, IL-1α, IL-1β, IL-6, IL-9, IL-12, IL-13, IL-17, IL-18, IL-22, IL-23. IL-24, IL-25, IL-26, IL-27, IL-33, IL-35, IL-2, IL-4, IL-5, IL-7, IL-10, IL-15, IL 41 or 40, which encodes a cytokine selected from -21, IL-25, IL-1RA, IFNα, IFNβ, IFNγ, TNFα, lymphotoxin α (LTA), Flt3L, GM-CSF and IL-8 The adenovirus described.   42. At least one further transgene encodes a chemokine selected from, for example, CCL2, CCL3, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19 and CCL21. The adenovirus according to any one of the above.   43. The adenovirus according to any one of claims 40 to 42, wherein the adenovirus comprises the sequence shown in SEQ ID NO: 101, 102, 103, or 298.   44. A composition comprising the adenovirus according to any one of claims 1-43 and a diluent or carrier.   45. The composition of claim 44, wherein the formulation comprises a second oncolytic virus, such as Ad11 or a derivative thereof, such as EnAd, in particular additional viruses are according to the present disclosure.   45. A method of treating a patient comprising administering a therapeutically effective amount of an adenovirus according to any one of claims 1-43 or a composition according to claim 44 or 45.   47. A method according to claim 46 for the treatment of cancer, in particular solid tumors.